Dissertations / Theses on the topic 'Oral Submucous Fibrosis (OSMF)'

Background: Oral submucous fibrosis (OSMF) is a pre-neoplastic condition, causally linked to areca nut consumption, the pathogenesis of which is poorly understood. TGF-β and disturbances in the balance between MMPs and TIMPs have been implicated in increased collagen deposition and fibrosis but no previous study has addressed the role of mesenchymal senescence in OSMF. Materials & Methods: Senescence and its secretome, DNA damage, oxidative damage, ROS production and mitochondrial damage were studied in OSMF in vivo and in vitro using ELISA, immunofluorescence, western blot and FACS techniques. Results: Senescent cells increased in all OSMF samples (1.9±0.3; p=0.004) peaking when dysplasia was present in the OSMF epithelium. There was increased oxidative damage (6.7±1.8; p=0.004); elevated DSBs (10.4±1.1; p=0.004) and P16ink4a accumulation (4.2±1.7; p=0.004). The results were similar in vitro. The OSMF fibroblasts demonstrated a reduced replicative lifespan (MPD-22±7.2; p=0.0001), despite having normal telomere lengths and in vivo growth rates. However, the OSMF fibroblasts showed increased ROS production and increased mitochondrial density and hyperpolarization, suggesting mitochondrial damage. The antioxidant, N-tert-Butyl-α- phenylnitrone (PBN) reduced the frequency of senescent fibroblasts and their associated markers in both OSMF and the control cultures. The mild uncoupling of mitochondrial oxidative phosphorylation from ROS generation with dinitrophenol (DNP) gave similar results. Cytokine profiles from cells obtained from OSMF showed a significant elevation of TIMP-1 (2217.4±406.5; p=0.003) and TIMP-2 (1763.2+/-363.7 pg/ml; p=0.004) production as compared to normal. The levels of MMP-1 (7.0±2.2 ng/ml; p=0.30), MMP-2 (157.8±91.3 ng/ml; p=0.75) and TGF-β1 (389.5±250.6 ng/ml; p=0.22) were not different to the normal and non-diseased controls (ND) collagen production was not elevated in OSMF in vitro (20.6±4.4 μg/ml; p=0.22). 3 Conclusion: Senescence, DNA damage, oxidative damage and P16inka accumulation are associated with neoplastic progression of OSMF. Elevated TIMP levels did not result in increased collagen secretion but may be an early marker of fibroblast aging and senescence.

Oral Potentially Malignant Disorders (OPMDs) are those oral mucosal conditions that have a predilection for becoming malignant (Warnakulasuriya et al. 2007). Similar to oral cancer itself and other oral conditions, OPMDs can cause significant morbidity that affects physical, social and psychological wellbeing, thus affecting the quality of life (QoL) of those so afflicted. The literature on the assessment of QoL in patients with OPMD is very limited, especially from developing countries, which might be due to the unavailability of a disease-specific QoL instrument (Tadakamadla et al. 2015). This thesis developed and validated a disease-specific self-administered QoL questionnaire for OPMD patients. The study was conducted in two phases in the oral medicine clinics of Panineeya Institute of Dental Sciences and Research Centre, Hyderabad, India. The first phase involved qualitative methodology for developing the OPMDQoL questionnaire. Qualitative data were collected through interviews and focus group discussions with 32 Telugu speaking patients with an OPMD who were undergoing treatment. Patients were those diagnosed with Oral Leukoplakia (OL), Oral Submucous Fibrosis (OSF) and Oral Lichen Planus (OLP) which are the most common OPMDs in the Indian subcontinent. Thematic analysis and coding of the qualitative data were conducted using Nvivo by two individuals independently to generate themes and the items for the questionnaire. Moreover, existing oral health-related QoL questionnaires, including those extant for patients with head and neck cancer, were reviewed to generate the items. A final list of 48 items was prepared, after inputs from oral physicians using a modified Delphi technique, from the 60 items that were generated through qualitative analysis and questionnaires. This was followed by the development of final questionnaire by item reduction using a clinical impact method which involved administration of the list of questions to 15 patients who rated the importance of each item. Based on these importance ratings, the final questionnaire (OPMDQoL) was developed: This consisted of 20 items under four domains: ‘difficulties with diagnosis’, ‘physical impairment and functional limitations’, ‘psychological and social wellbeing’ and the ‘effect of treatment on daily life’. Each item is scored using a five-point Likert scale, and the overall OPMDQoL and domain scores are calculated by summing the scores of each item with a higher score representing poorer QoL. In the second phase, the validity and reliability of OPMDQoL was assessed. OPMDQoL, Telugu translations of the Chronic Oral Mucosal Disease Questionnaire (COMDQ) (Ni Riordain et al. 2011b) and Short form 12 item (version 2) of the health survey questionnaire (SF-12v2) (Ware et al. 1996) were administered to 150 OPMD patients (50 each of OL, OSF and OLP) and to controls who were free from any type of OPMD. A thorough clinical examination was conducted and history, including lifestyle habits, was recorded from all patients. OPMDQoL demonstrated good discriminant validity as patients presented poorer scores than the controls. Also, it had good convergent validity evaluated by assessing its correlation with an existing questionnaire (COMDQ) designed for use in patients with any type of oral mucosal disease. An exploratory factor analysis was also conducted, and findings demonstrated a four-factor structure that conforms to the hypothesised four domains. The impact of the clinical diagnosis (OL, OSF or OLP) and of disease severity on disease-specific and generic QoL was then assessed. Patients with OL reported better OPMDQoL than those with OSF and OLP, while no differences were observed between the three OPMDs for the mental health components of the SF-12v2 questionnaire and for the ‘psychological and functional wellbeing’ domain of the OPMDQoL instrument. When domain scores were compared, OLP patients reported significantly higher scores for the domain ‘difficulties with diagnosis’ while OL patients presented lower scores for the dimension, ‘physical impairment and functional limitations’ than did patients with the other OPMD. Disease severity had a significant association with both OPMDQoL and with the mental health component of SF-12v2, QoL diminishing as disease severity increased. In conclusion, the newly developed OPMDQoL exhibited good validity and reliability in a sample of Telugu Speaking OPMD patients. Disease severity was found to be associated with poor QoL assessed using OPMDQoL. Taken together, the results of this work indicate that this questionnaire might be used in studies of disease progression and to evaluate response to therapy, in routine clinical practice. OPMDQoL can be used in different cultures and languages following cross-cultural adaptation and can also be used to compare the effectiveness of treatments.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Dentistry&Oral Hlth
Griffith Health
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The pathogenesis of oral submucous fibrosis (OSMF) is due to a complex interplay between the production and degradation of extracellular matrix (ECM) protein components. In tissue fibrosis, there is a net accumulation of collagen as a result of an imbalance between enhanced production, deposition and impaired degradation of ECM components. OSMF is a chronic inflammatory condition of the oral cavity and regulation of a number of pro-inflammatory and fibrogenic cytokines such as interleukine-1, -6 and -8 isoforms, TGF-β, PDGF, bFGF, IFN-γ and TNF-α has been reported in OSMF tissues. The expression of these growth factors has a bearing on the epithelial changes as well as proliferation and differentiation of oral fibroblasts into ECM protein producing myofibroblast cells. One key modulator of fibrosis in several organs has been TGF-β. Overproduction of TGF-β mRNA and protein has been reported in several fibrotic disorders including that of skin, lungs, liver, kidney and heart. This is mainly due to stimulation of ECM genes by TGF-β. Although there have been few reports suggesting the over production of TGF-β in OSMF tissues, the specific isoforms involved or the mechanisms are poorly understood. Areca nut components, especially arecoline have been implicated in the pathophysiology of OSMF. Few reports indicate the involvement of arecoline in the regulation of collagen production and activity of collagenases and their inhibitors in oral fibroblast cells. Moreover, the alkaloid is involved in initiating epithelial inflammation by inducing COX-2, prostaglandin E2, IL-1α, IL-6 and IL-8 in KB oral carcinoma cells and oral fibroblast cells. These and other reports strongly suggest that changes in gene expression mediated by Arecoline may be central to the progression of OSMF. Not much is known about arecoline-mediated cellular signaling events except for few recent reports that suggest the activation of MAPK pathways. In neuronal and colonic smooth muscle cells of mouse, rat and rabbit, the actions of Arecoline have been reported to be through the activation of muscarinic acetylcholine receptors. Direct binding of arecoline to human M1, 2 and 3 muscarinic receptor isoforms have been shown in brain tissues. Stimulation of these receptors alters the intracellular levels of Ca+2 and cAMP, which are important second messengers. The cholinergic potential of arecoline may be important for their roles in arecoline-mediated signaling events. The expression of muscarinic acetylcholine receptors has been reported in several cell types besides neuronal and excitatory cells. Although several gene expression changes have been reported following Arecoline treatment of a variety of cells, the mechanism of such regulations is not established. Hence in order to understand the role of arecoline in OSMF disease process, we undertook studies that provide insights into arecoline action in epithelial and fibroblast cells and possible molecular mechanisms. The objectives are to study: 1. The role of arecoline in cellular proliferation, cell-cycle regulation and apoptosis in human normal keratinocytes. 2. Mechanism of regulation of gene expression by arecoline in normal keratinocytes. 3. Mechanism of regulation of gene expression by arecoline in human normal oral fibroblasts. In order to achieve the above objectives, a human keratinocyte cell line, HaCaT and an oral periodontal fibroblast cell line (PDC) were utilized. The cells were treated with arecoline and a variety of assays including RT-PCR analysis of mRNA of several genes, phosphorylation status of MAPK pathway intermediates, cell cycle analysis and other cellular and molecular methods have been employed. Following arecoline treatment, there is induction of oxidative stress, growth arrest and epithelial cell death. Since actions of TGF-β are central to most fibrotic disorders and arecoline has been implicated in OSMF, it is hypothesized that arecoline may influence fibrosis via TGF-β pathway. Towards this, several TGF-β target genes that may have a possible role in fibrosis have been studied in arecoline treated epithelial and fibroblast cells. Since arecoline mediated oxidative stress has been reported, the regulation of genes that are involved in stress response pathway have been studied for induction by arecoline in epithelial cells. The results presented in this thesis suggest the up regulation of oxidative stress-responsive genes in HaCaT cells including HOX-1, FTL, G6PD, GCLC and GRD in HaCaT cells. Oxidative stress is a major inducer of inflammatory response in the epithelial tissues. The expression of IL-1α, an important inflammatory cytokine is induced by arecoline in HaCaT cells in response to oxidative stress via the activation of p38 MAPK pathway. Interestingly, activation of MAPK pathways by arecoline is involved in the regulation of common target genes of arecoline and TGF-β and also in the induction of TGF-β−responsive promoter reporter construct, p3TP-lux activity in HaCaT cells. Due to the involvement of TGF-β in fibrosis, regulation of TGF-β pathway genes by arecoline has been studied both in HaCaT and PDC cells. In HaCaT cells, arecoline induces the expression of TGF-β2 mRNA while TβRII expression is down regulated. The expression of the rest of TGF-β/SMAD pathway genes including TGF-β1, β3, TβRI, SMAD1, 2, 3, 4 and 7 are not affected by arecoline in HaCaT cells. Over expression of TGF-β2 is also observed in most of the OSMF tissues compared to normal oral tissues. However, in normal oral fibroblast cells, we observed that the TGF-β/SMAD pathway genes are not regulated by arecoline. These results suggest the possible involvement of arecoline-mediated induction of TGF-β2 in epithelial cells in OSMF disease development. We investigated the signaling pathways involved in the regulation of TGF-β2 and found that stimulation of M3 muscarinic receptor by arecoline leads to the induction of TGF-β2 expression in HaCaT cells via PKC pathway. TGM-2 is an important TGF-β target gene involved in the cross linking of ECM proteins. Arecoline-mediated induction of TGM2 mRNA and transglutaminase activity are observed in oral fibroblast cells, PDC. The induction of TGM-2 was found to be independent of oxidative stress and TGF-β, but dependent on muscarinic acid receptor activation by arecoline and involves cytosolic cAMP. When tested in OSMF tissues, there was an increased expression of TGF-β2, TSP1 and TGM2 as compared to normal tissues suggesting a possible role of these genes in arecoline-mediated progression of OSMF. Interleukin-8 (IL-8), which is involved in inflammation has been reported to be regulated by TGF-β in a cell type specific manner. In several cell types including human endometrial stromal cells, LnCaP (prostate cancer cells), human retinal pigment epithelial cells and rat lung alveolar epithelial (LM5) cells etc., TGF-β up regulates the expression of IL-8 mRNA. Arecoline was found to down regulate IL-8 expression in PDC cells as measured by RT-PCR. Interestingly, the presence of serum along with arecoline induces the expression of IL-8 in PDC cells suggesting the modulation of arecoline-mediated gene regulation by a serum activated signaling pathway. Intriguingly, arecoline treatment led to down regulation of collagens in PDC cells. However, collagen genes are induced in PDC cells in the presence of HaCaT spent medium by arecoline suggesting a role for factor(s) secreted by epithelial cells in the regulation of collagen genes by arecoline. This factor could be an isoform of TGF-β as shown by blocking the induction of collagens by the TGF-β inhibitor, βLAP. Taken together, all these results indicate the ability of arecoline to cause fibrosis in a tissue environment where both epithelial and fibroblasts respond to arecoline and mutually contribute to the disease manifestation. Major conclusions from this study includes, 1] cell death in epithelial cells due to oxidative stress following arecoline treatment, 2] regulation of gene expression by arecoline involves MAPK, PKC pathways, 3] muscarinic acid receptor and oxidative stress are also important for regulation gene expression by arecoline. The most important inference from this study is the possible paracrine influence of TGF-β isoforms secreted by epithelial cells on the oral fibroblasts in determining the progression of OSMF. In summary, in this thesis, an attempt has been made to study the molecular mechanisms and role of arecoline, an alkaloid in conferring gene expression changes that may lead to the initiation and progression of oral sub mucous fibrosis.

The pathogenesis of oral submucous fibrosis (OSMF) is due to a complex interplay between the production and degradation of extracellular matrix (ECM) protein components. In tissue fibrosis, there is a net accumulation of collagen as a result of an imbalance between enhanced production, deposition and impaired degradation of ECM components. OSMF is a chronic inflammatory condition of the oral cavity and regulation of a number of pro-inflammatory and fibrogenic cytokines such as interleukine-1, -6 and -8 isoforms, TGF-β, PDGF, bFGF, IFN-γ and TNF-α has been reported in OSMF tissues. The expression of these growth factors has a bearing on the epithelial changes as well as proliferation and differentiation of oral fibroblasts into ECM protein producing myofibroblast cells. One key modulator of fibrosis in several organs has been TGF-β. Overproduction of TGF-β mRNA and protein has been reported in several fibrotic disorders including that of skin, lungs, liver, kidney and heart. This is mainly due to stimulation of ECM genes by TGF-β. Although there have been few reports suggesting the over production of TGF-β in OSMF tissues, the specific isoforms involved or the mechanisms are poorly understood. Areca nut components, especially arecoline have been implicated in the pathophysiology of OSMF. Few reports indicate the involvement of arecoline in the regulation of collagen production and activity of collagenases and their inhibitors in oral fibroblast cells. Moreover, the alkaloid is involved in initiating epithelial inflammation by inducing COX-2, prostaglandin E2, IL-1α, IL-6 and IL-8 in KB oral carcinoma cells and oral fibroblast cells. These and other reports strongly suggest that changes in gene expression mediated by Arecoline may be central to the progression of OSMF. Not much is known about arecoline-mediated cellular signaling events except for few recent reports that suggest the activation of MAPK pathways. In neuronal and colonic smooth muscle cells of mouse, rat and rabbit, the actions of Arecoline have been reported to be through the activation of muscarinic acetylcholine receptors. Direct binding of arecoline to human M1, 2 and 3 muscarinic receptor isoforms have been shown in brain tissues. Stimulation of these receptors alters the intracellular levels of Ca+2 and cAMP, which are important second messengers. The cholinergic potential of arecoline may be important for their roles in arecoline-mediated signaling events. The expression of muscarinic acetylcholine receptors has been reported in several cell types besides neuronal and excitatory cells. Although several gene expression changes have been reported following Arecoline treatment of a variety of cells, the mechanism of such regulations is not established. Hence in order to understand the role of arecoline in OSMF disease process, we undertook studies that provide insights into arecoline action in epithelial and fibroblast cells and possible molecular mechanisms. The objectives are to study: 1. The role of arecoline in cellular proliferation, cell-cycle regulation and apoptosis in human normal keratinocytes. 2. Mechanism of regulation of gene expression by arecoline in normal keratinocytes. 3. Mechanism of regulation of gene expression by arecoline in human normal oral fibroblasts. In order to achieve the above objectives, a human keratinocyte cell line, HaCaT and an oral periodontal fibroblast cell line (PDC) were utilized. The cells were treated with arecoline and a variety of assays including RT-PCR analysis of mRNA of several genes, phosphorylation status of MAPK pathway intermediates, cell cycle analysis and other cellular and molecular methods have been employed. Following arecoline treatment, there is induction of oxidative stress, growth arrest and epithelial cell death. Since actions of TGF-β are central to most fibrotic disorders and arecoline has been implicated in OSMF, it is hypothesized that arecoline may influence fibrosis via TGF-β pathway. Towards this, several TGF-β target genes that may have a possible role in fibrosis have been studied in arecoline treated epithelial and fibroblast cells. Since arecoline mediated oxidative stress has been reported, the regulation of genes that are involved in stress response pathway have been studied for induction by arecoline in epithelial cells. The results presented in this thesis suggest the up regulation of oxidative stress-responsive genes in HaCaT cells including HOX-1, FTL, G6PD, GCLC and GRD in HaCaT cells. Oxidative stress is a major inducer of inflammatory response in the epithelial tissues. The expression of IL-1α, an important inflammatory cytokine is induced by arecoline in HaCaT cells in response to oxidative stress via the activation of p38 MAPK pathway. Interestingly, activation of MAPK pathways by arecoline is involved in the regulation of common target genes of arecoline and TGF-β and also in the induction of TGF-β−responsive promoter reporter construct, p3TP-lux activity in HaCaT cells. Due to the involvement of TGF-β in fibrosis, regulation of TGF-β pathway genes by arecoline has been studied both in HaCaT and PDC cells. In HaCaT cells, arecoline induces the expression of TGF-β2 mRNA while TβRII expression is down regulated. The expression of the rest of TGF-β/SMAD pathway genes including TGF-β1, β3, TβRI, SMAD1, 2, 3, 4 and 7 are not affected by arecoline in HaCaT cells. Over expression of TGF-β2 is also observed in most of the OSMF tissues compared to normal oral tissues. However, in normal oral fibroblast cells, we observed that the TGF-β/SMAD pathway genes are not regulated by arecoline. These results suggest the possible involvement of arecoline-mediated induction of TGF-β2 in epithelial cells in OSMF disease development. We investigated the signaling pathways involved in the regulation of TGF-β2 and found that stimulation of M3 muscarinic receptor by arecoline leads to the induction of TGF-β2 expression in HaCaT cells via PKC pathway. TGM-2 is an important TGF-β target gene involved in the cross linking of ECM proteins. Arecoline-mediated induction of TGM2 mRNA and transglutaminase activity are observed in oral fibroblast cells, PDC. The induction of TGM-2 was found to be independent of oxidative stress and TGF-β, but dependent on muscarinic acid receptor activation by arecoline and involves cytosolic cAMP. When tested in OSMF tissues, there was an increased expression of TGF-β2, TSP1 and TGM2 as compared to normal tissues suggesting a possible role of these genes in arecoline-mediated progression of OSMF. Interleukin-8 (IL-8), which is involved in inflammation has been reported to be regulated by TGF-β in a cell type specific manner. In several cell types including human endometrial stromal cells, LnCaP (prostate cancer cells), human retinal pigment epithelial cells and rat lung alveolar epithelial (LM5) cells etc., TGF-β up regulates the expression of IL-8 mRNA. Arecoline was found to down regulate IL-8 expression in PDC cells as measured by RT-PCR. Interestingly, the presence of serum along with arecoline induces the expression of IL-8 in PDC cells suggesting the modulation of arecoline-mediated gene regulation by a serum activated signaling pathway. Intriguingly, arecoline treatment led to down regulation of collagens in PDC cells. However, collagen genes are induced in PDC cells in the presence of HaCaT spent medium by arecoline suggesting a role for factor(s) secreted by epithelial cells in the regulation of collagen genes by arecoline. This factor could be an isoform of TGF-β as shown by blocking the induction of collagens by the TGF-β inhibitor, βLAP. Taken together, all these results indicate the ability of arecoline to cause fibrosis in a tissue environment where both epithelial and fibroblasts respond to arecoline and mutually contribute to the disease manifestation. Major conclusions from this study includes, 1] cell death in epithelial cells due to oxidative stress following arecoline treatment, 2] regulation of gene expression by arecoline involves MAPK, PKC pathways, 3] muscarinic acid receptor and oxidative stress are also important for regulation gene expression by arecoline. The most important inference from this study is the possible paracrine influence of TGF-β isoforms secreted by epithelial cells on the oral fibroblasts in determining the progression of OSMF. In summary, in this thesis, an attempt has been made to study the molecular mechanisms and role of arecoline, an alkaloid in conferring gene expression changes that may lead to the initiation and progression of oral sub mucous fibrosis.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.

Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.

Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.

博士
中山醫學大學
生化暨生物科技研究所
93
Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of matrix metalloproteinases (MMPs) and plasminogen activator (PA)/plasmin system in the pathogenesis of OSF. In the present study, we examined the activity of TIMP-1 and PAI-1 from cell cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 and PAI-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP-1 or MMP-2 production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 and PAI-1 expression at the concentration level under 20 ug/ml in a dose-dependent manner. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production in a dose-dependent manner. In this study, we also investigated the genetic analysis of PAI-1 in the promoter region between OSF and normal buccal mucosa. PAI-1 genotyping with allele specific polymerase chain reaction (ASPCR) and allele-specific restriction enzyme site analysis (ASRS) was performed in the tissue of 52 OSF and 32 normal buccal mucosa. There were significant differences between the OSF and BMF for the frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes (P < 0.05). In the OSF group, it had a hight frequency of PAI-1 (4G/4G) genotypes than those in BMF group (P < 0.05) Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1、t-PA and PAI-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF. Furthermore, our finding also suggested that the distribution pattern of PAI-1 promoter were different between OSF and BMF tissue. PAI-1 4G allele, with a higher transcription activity, was more prevalent in OSF.

碩士
台北醫學院
口腔復健醫學研究所
90
Oral submucous fibrosis（OSF）is characterized by symptoms such as intolerance to spicy food, burning seasation, altered salivation, progressive difficulty in opening the mouth. The purpose of the present investigation is to describe the ultrastructural findings（TEM ＆ SEM）of the oral epithelium , subepithelial connective tissue, muscle, vessels and nerve of OSF. Biopsies were taken from the buccal mucosa of 20 cases of typical OSF. Routine EM procedures were done and sections were examined by TEM and SEM.The epithelium showed degeneration. The intercellular spaces of cells were edematous. The basal lamina appeared branched, thickened and duplicated in almost all the lesions. The cells showed pleomorphism. There were hylinized homogenous granules in the connective tissue. The collagen fibers of OSF tissue were excessive and fine (immature).The muscle was involved as well as submucous layer in OSF. Muscle fibers underwent degeneration and plasma membrane was vacuolization and hyalinization. The endothelial cells of vessels in muscle showed degeneration. Peripheral nerve also showed degeneration. The basal laminae on each side of perineural cells were thickened and incomplete. Microfibrils were compressed and scattered at the periphery of the nerve. Endoneural collagen fibrils were also compacted and concentrated around nerve fibers. The Schwann cell sheaths were invested with remarkably thickened basal lamina. The axons appeared degenerated with swollen mitochondria and hyalinized cytoplasm. The hyalinized axons were frequently observed at the distal portion of nerves. The disorganized changes of neural tissues in OSF were not previously reported and might be one of the factors resulting in the intolerance to spicy food and the burning sensation of the mouth.

博士
國立臺灣大學
流行病學研究所
89
Abstract Oral submucous fibrosis (OSF) is a chronic disease of oral mucosa. Its main characteristics is the abnormal accumulation of submucous collagen. OSF is also an oral premalignancy. According to several long term follow-up studies, 2.3%~33.3% OSF will progress into oral cancer. The etiology of OSF remains unclear. Most studies suggest betel quid chewing is an important risk factor for OSF, but only a small proportion of betel quid chewers get OSF. This imply that genetic susceptibility may also play an important role in the etiology of OSF. This project has two aims: 1) to examine associations with OSF for genetic polymorphisms of collagenase gene, cystatin gene, etc. 2) to compare risk factors for OSF and oral cancer. Study subjects was recruited from both hospital and communities. A total of 167 OSF cases, 111 healthy betel quid chewers, 203 non-chewers, and 77 oral cancer patients was recruited from the dental clinic of NTUH as our hospital subjects. We also recruited 173 healthy betel quid chewers and 105 non-chewers from Makung, Chutung, Potzu, Kaoshu and Sanchi as our community controls. Standardized personal interview-based on structure questionnaire was carried out to obtain information on risk factors for OSF and oral cancer. Polymerase chain reaction and restriction fragment length polymorphism was used to genotype the genes of collagen and collagenases. Multiple logistic regression analysis was used to estimate mutivariate-adjusted odds ratios with their 95% confidence intervals of various risk factors. Among the ten candidate gene loci, a biallelic promotor-region polymorphism of the TNFA gene at position —308 was found to have association with OSF. As previous study demonstrated the rare allele, TNF*2, having increased promotor function compared with the common allele, TNF*1 and the transcriptional induction of TNF-α in collagenase, we found the *1/*1 genotype is more risky with an odds ratio of 2.42 (95% CI=1.32-4.44, p=0.0045). This result is in keeping with a protective role of TNF-α against OSF. We also found the importance of smoking for betel quid chewers progressing into oral cancers and the probible role of alcohol drinking for OSF patients progressing into oral cancers. Moreover, the association between OSF and TNFA gene mentioned before was found to be independent of oral cancer.

碩士
國立成功大學
口腔醫學研究所
102
Oral submucous fibrosis (OSF) is a premalignant condition which is related to oral cancer formation. Concept of field cancerization fit the clinical outcome of various kinds of malignant tumor such as oral cancer, breast cancer, and hepatocellular carcinoma. Carcinogen-exposed tissue fields develop clonal malignant cells during evolution of neoplastic lesions. But the mechanism of field cancerization was not well understood. Cancer as a disease of unbalancing epithelial–mesenchymal interactions and extracellular matrix regulation was well accepted. Tumor microenvironment and its stromal cell had been convinced to play an important role on carcinogenesis, metastasis and drug resistance. In recent year, many researchers proposed that field cancerization was determinant primarily by the stroma, but it lack human tissue evidence. OSF is a stromal disease, and related to cancer formation. Therefore, we would like to determine whether OSF cases were highly associated with multiple oral filed tumorgenesis. We survey OSF cases in NCKU hospital, and higher incidence of multiple primary oral cancer was noted (P〈0.001). Conditioned medium collected from primary cultured OSF fibroblast could promote oral precancer cell (DOK) and oral cancer cell migration just like that from cancer associated fibroblast. Fibronectin, the cell migration related ECM was highly expressed in tumor and OSF stroma. Taken together, our result indicated that benign precancer lesions such as OSF stroma can have similar phenotype with malignant stroma which may provide a possible model to explain stroma is a primary determinant of multifocal epithelial tumor and field cancerization. We hope that our study could provide some clue for prevention of primary or second primary tumor formation.

博士
國立臺灣大學
臨床牙醫學研究所
91
In Taiwan，the increasing areca quid (AQ) chewing population and motality rate of oral cancer are still the critical problems of public health. Among AQ chewing-related diseases, oral submucous fibrosis (OSF) is one of the most common oral diseses, whose pathogenesis is still not clear. In part I of this study, we studied the expression of COX-2 in OSF epithelium to elucidate whether prostaglandin-induced inflammation was related to the pathogenesis of OSF and studied the HLA typing of OSF patient to assess whether patients with particular HLA phenotypes and haplotypes were prone to have OSF. In part II, autofluorescence spectroscopy was used to characterize the special fluorescence spectrum of OSF, and to investigate the effect of OSF on other AQ chewing-related lesions like epithelial hyperkeratosis (EH), epithelial dysplasia (ED) and oral squamous cell carcinoma (SCC). In part III, the non-surgical treatment with submucosal injection of triamcinolone acetonide for OSF was evaluated. The results of this study showed the COX-2 positive rate of OSF is significantly higher than that of normal oral mucosa (NOM) (P=0.016). The higher exprssion of COX-2 was significantly correlated with the longer duration and the greater total amount of AQ chewing. The HLA typing for 135 OSF patients and 540 healthy controls showed that the phenotype frequency of HLA-B76 and the haplotype frequencies of HLA-B48/Cw7、 -B51/Cw7 and -B62/Cw7 were significantly higher in OSF patients than in normal controls after correction of P value. This finding suggests that in Taiwan some specific HLA phenotypes and haplotypes might play important roles in genetic susceptibility of OSF. Autofluorescence spectroscopy has been used as a method to screen cancer, and there were many researches focusing on oral leukoplakia and oral cancer, but none was done on OSF. Our findings showed the unique autofluorescence spectrum of OSF in which the 380-nm emission peak was higher in OSF than in EH, ED, and SCC, but the 460-nm emission peak was lower in OSF than in EH, ED and SCC. We also found the effect of OSF on the autofluorescence spectrum of EH, ED and SCC, which resulted in the unreliable autofluorescence spectra for these lesions. When OSF patients were treated by submucosal injection of traamcinolone acetonide, our data showed that the significant improvement of MMO was dependent on on forced mouth opening but not on the steroid used. . According to the findings of our study, there are many works that could be done in the future. These include the further investigations of the participation of inflammation mechanism in OSF pathogenesis, of the application of microarry and single neucleotide polymorphism on the genetic susceptibility of OSF, of the application of the d-aminolevulinic acid on the diagnosis and photodynamic therapy for AQ chewing-related diseases, of the closed follow-up of the non-surgically treated OSF patients to confirm that there is no significant difference in the result of treatment with or without steroid and of the application of artificial skin on the treatmnet of OSF.

碩士
國立臺灣大學
臨床牙醫學研究所
89
In this study, we used an immunohistochemical method to study the expressions of apoptosis-associated proteins（BCL-2、BCL-XL、MCL-1、BAX、BAK and p53） in 70 cases of oral submucous fibrosis（OSF, experimental group）, 34 cases of normal oral mucosa（NOM, normal control group） and 69 cases of oral squamous cell carcinoma （OSCC, disease control group）. The expressions of apoptosis-associated proteins in OSFs were further correlated with the clinicopathological parameters of the OSF patients. The positive staining rates of BCL-2 in NOM, OSF and OSCC specimens were 0.0%, 4.3% and 4.3%, respectively. No statistically significant difference was found between the positive staining rates of BCL-2 in NOM, OSF and OSCC specimens（p=0.467）. There was a marginally significant correlation between the expression of BCL-2 in OSF epithelium and the patients’ daily consumption of the cigarettes (p=0.0526). If the patients’ daily consumption of the cigarettes increased one pack, then the odds in favor of the positive staining rate of the BCL-2 increased to 7.11 times. The positive staining rates of BCL-XL in NOM, OSF and OSCC specimens were 44.1%, 41.4% and 21.7%, respectively. The BCL-XL expression rate in OSCC specimens was significantly lower than that in NOM specimens（p<0.001, adds ratio=0.352）and that in OSF specimens（p=0.021, adds ratio=0.393）. There was no statistically significant correlation between the expression of the BCL-XL in OSF epithelium and clinicopathological parameters of the OSF patients. The positive staining rates of MCL-1 in NOM, OSF and OSCC specimens were 100%, 70% and 68.1%, respectively. The MCL-1 expression rate in NOM specimens was significantly greater than that in OSF specimens（p<0.001）and that in OSCC specimens（p<0.001）. A statistically significant correlation was found between the expression of MCL-1 in OSF epithelium and the patients’ daily consumption of betel quids (p=0.0137) or depth of connective tissue fibrosis（p=0.006）. If the fibrous depth increases one minimeter, then the odds in favor of the positive staining rate of MCL-1 decreased to 0.013 times. The positive staining rates of BAX in NOM, OSF and OSCC specimens were 47.1%, 92.9% and 87%, respectively. The BAX expression rate in OSF and OSCC specimens were significantly greater than that in NOM specimens（p<0.001）. There was a marginally significant correlation between the expression of BAX in OSF epithelium and the patients’ daily consumption of the cigarettes (p=0.1733). If the patients’ daily consumption of cigarettes increased one pack, then the odds in favor of the positive staining rate of the BAX increased to 4.714 times. The positive staining rates of BAK in NOM, OSF and OSCC specimens were 67.6%, 77.1% and 97.1%, respectively. The BAK expression rate in OSCC specimens was significantly greater than that in OSF specimens（p=0.01, adds ratio=9.926）and that in NOM specimens（p<0.001, adds ratio=16.022）. There was no statistically significant correlation between the expression of BAK in OSF epithelium and clinicopathological parameters of the OSF patients. The positive staining rates of p53 in NOM, OSF and OSCC specimens were 17.6%, 37.1% and 33.3%, respectively. The p53 expression rate in OSCC specimens or in OSF specimens was significantly greater than that in NOM specimens. The long-term exposure of OSF epithelial cells to carcinogenic ingredients contained in betel quid and tobacco smoke caused DNA damage. This DNA damage induced the production of wild-type p53 proteins which in turn induced the expression of pro-apoptotic proteins (BAX, BAK) in OSF epithelial cells. These pro-apoptotic proteins further caused the apoptosis of OSF epithelial cells, resulting in atrophy of the OSF epithelium. In addition, the wild-type p53 proteins also inhibited the expression of BCL-2, resulting in very low production of BCL-2 in OSF epithelial cells. In OSF epithelium, the expression rate of MCL-1 was higher than that of BCL-2 and that of BCL-XL. Therefore, MCL-1 may be more important than BCL-2 and BCL-XL as anti-apoptotic protein in OSF epithelial cells.

博士
中山醫學大學
牙醫學系博士班
103
Oral submucous fibrosis (OSF), a chronic progressive scarring disease, has been considered as a pre-cancerous condition of oral mucosa. In the study, we investigated the functional role of Twist, an epithelial-mesenchymal transition (EMT) transcriptional factor, in myofibroblastic differentiation activity of OSF. Arecoline, a major areca nut alkaloid, was used to explore whether expression of Twist could be changed dose-dependently in human primary buccal mucosal fibroblasts (BMFs). Collagen gel contraction and migration capability in arecoline-stimulated BMFs and primary oral submucous fibrosis-derived fibroblasts (OSFs) with Twist knockdown was presented. We observed that the treatment of arecoline dose-dependently increased twist expression transcript and protein levels in BMFs. The myofibroblast activity including collagen gel contraction and migration capability also induced by arecoline, while knockdown of Twist reversed these phenomena. Importantly, inhibition of Twist led to the suppression collagen contraction and wound healing capability of primary cultivated OSFs. Clinically, Twist transcript and protein expression was higher expression in areca quid chewing-associated OSF tissues than in normal oral mucosa tissues. These evidences suggest that up-regulation of Twist might be involved in the pathogenesis of areca quid-associated OSF through dysregulation of myofibroblast activity.

碩士
中山醫學大學
生物醫學科學學系碩士班
101
There are more than two million Taiwanese with betel nut consumption habit. This behavior causes several oral diseases including oral submucous fibrosis (OSF) and oral cancer but the pathogenesis of betel nut induced OSF is still not fully understood. Myofibroblasts, which identified as alpha-smooth muscle actin (α-SMA) positive cells, are frequently found in fibrotic tissues and participate in fibrogenic process through collagen secretion and induction of tissue contraction. Arecoline is the well-known alkaloid natural product in betel nut and has been suggested to induce OSF and oral cancer. Here we investigated the role of arecoline in the differentiation of myofibroblasts from normal buccal mucosal fibroblasts (BMFs). We first determined the effect of arecoline in cell growth of firbolasts and the IC50 is 72.4±27.9 μg/ml. We further observed that arecoline could increase cell proliferation in BMFs at the concentration of 10-20μg/ml and induce the expression of α-SMA and vimentin, a marker of mesenchymal cells, simultaneously. In order to understand the molecular mechanisms of arecoline-induced α-SMA expression, we focused on the molecules involving in epithelial-mesenchymal transition (EMT). By western blot, we found that the transcriptional repressor in EMT process, Zinc finger E-box binding homeobox 1 (ZEB-1), were induced by arecoline simultaneously with α-SMA expression in normal BMFs. Through collagen contraction assay, we identified that arecoline induced myofibroblast activity in normal BMFs. Arecoline also could increase α-SMA expression in normal BMFs through ZEB-1 by directly binding to E-box domain of α-SMA promoter. By RNA interference, we further demonstrated that knockdown of ZEB-1 could decrease arecoline-induced α-SMA expression, as well as the collagen contraction in normal BMFs. Understanding the role of ZEB-1 in arecoline-induced OSF may provide a new insight in the new drug development for treating OSF disease.

碩士
國立臺灣大學
口腔生物科學研究所
98
Oral submucous fibrosis (OSF) is a chronic and inflammatory disease of oral mucous. Epidemiological evidences strongly indicate the association of chewing areca quid habit and OSF which is a kind of potentially malignant disorders. Many evidences have showed that α-smooth muscle actin (α-SMA) is overexpressed in some organs, such as lung, liver, and skin, but the α-SMA expression in OSF is not clear, therefore, in this study, we investigated the correlation of α-SMA expression and the pathogenesis in OSF by immunohistochemistry (IHC). The result shows that the expression of α-SMA in normal buccal mucosa only exists in the smooth muscle cells (positive control) of blood vessels, however, α-SMA is overexpressed in OSF. For detecting the effect of arecoline and TGF-β in OSF we treated normal oral mucous fibroblasts (NOM) with above components, and then analyzed the expression of α-SMA by Western Blot. The result showed that both arecoline and TGF-β were able to induce the expression of α-SMA in the dose-dependent manner. Finally, we treated NOM with Curcumin, Epigallocatechin-3-gallate (EGCG), or Trichostatin A (TSA), the result showed that all of them were able to reduce the expression of α-SMA induced by arecoline or TGF-β. In our study, we found that OSF expressed much more α-SMA than NOM. Besides, both arecoline and TGF-β can induce the expression of α-SMA. The induced expression of α-SMA by arecoline or TGF-β can be reduced by Curcumin, EGCG, or TSA. Keywords: oral submucous fibrosis, OSF, α-SMA

碩士
長庚大學
顱顏口腔醫學研究所
98
Background and purpose : Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by progressive fibrosis of oral connective tissues and accumulation of collagen in the propria of oral mucosa. It is thought to be an oral precancerous condition with about 7.6% malignant transformation rates. However, there is short of molecular markers that can predict the outcome of OSF transformation. It is of critical importance to develop a methodology that can provide the means to stratify the potential risk of precursor lesions. Materials and methods : In order to globally survey the altered gene expressions in OSF patients, Affymetrix microarrays were used for transcriptome profiling between 21 normal mucosa samples and 6 OSF samples. After analyzing the microarray data by MetaCoreTM algorithm, five candidate genes including MMP1, KRT17, ENO3, KBTBD10, MYH2 were selected for further validated in normal, OSF and oral cancer tissues by RT-PCR analysis. Results : Total of 694 genes were differentially expressed at least 2-fold in OSF patients, including 397 up-regulated and 297 down-regulated. Several regulatory pathways were found significant (p&lt;0.001), including small GTPase signal transduction (p=4.12E-05), transcription regulation (p = 6.81E-05) and cell adhesion regulation (p = 4.41E-04). Compared to normal mucosa samples, KBTBD10 (p=0.006) was significant up-regulated in OSF tissues and in tumors (p=0.014). Similarly, ENO3 (p=0.004) was significant up-regulated in OSF tissues and in tumors (p=0.014). MYH2 did not show any statistical differences between normal mucosa and OSF groups (p=0.071) or normal and tumor groups (p=0.11). KRT17 (p=0.03) and MMP1 (p&lt;0.001) show statistical difference between OSF and tumor. Conclusions : These results indicated that ENO3 and KBTBD10 play early roles during oral carcinogenesis, especially in OSF formation. MMP1 and KRT17 play later roles in oral cancer development. This study may be use in clinical applications in the future.

碩士
國立臺灣大學
口腔生物科學研究所
100
Oral submucous fibrosis is a continuous, chronic, insidious and inflammatory disease of oral mucous that is a kind of potentially malignant disorders. OSF is mainly due to consumed areca quid which is lead to fibrosis in the oral cavity. In the progression of inflammatory, some pro-inflammatory and pro-fibrotic cytokine could be upregulated, like TGF-β、IFN-γ, etc., the exact pathogenesis of OSF and cytokine was still unclear and needed to study .However, the previously study had showed that B7-H1 was regulated by IFN-γ in the human dermal fibroblast. Therefore, in this study, we investigated whether B7-H1 was involved in the mechanisms of OSF diseases. First, we investigated the expression of B7-H1 in normal oral mucosa tissue and in OSF patient’s tissue by immunohistochemistry (IHC).The result showed that most of fibroblasts that express B7-H1 was close to epithelium layer in OSF tissue. It also showed that the expression of B7-H1 in epithelium layer in OSF is much more than in NOM. Then, we treated NOM fibroblast with arecoline and analyzed the expression of B7-H1 by Western blot. The result showed that B7-H1 is up-regulated by arecoline in NOM, and JNK inhibitor could reduce B7-H1 expression. The reference indicated that B7-H1 over-expression could be regulated Epithelial-mesenchymal transition (EMT) in human skin cancer. The result of IHC showed that fibroblasts that express B7-H1 were in the juxta-epithelial connective tissue. The reference showed that EMT is one of important mechanisms of OSF, and to investigate whether B7-H1 is involved in EMT which is lead to fibrosis. We treated S-G epithelial cell with TGF-β, and the result showed that EMT biomarker protein and B7-H1 expression was increased. And then, we pre-treat S-G cell with JNK inhibitor and then treat with TGF-β, the result showed that the expression of B7-H1 and some EMT biomarker both were reduced. And then, we used B7-H1 specific siRNA to knockdown its expression in S-G cells. The result showed that knockdown of expression of B7-H1 influenced EMT biomarker gene expression.

碩士
高雄醫學大學
藥學系臨床藥學碩士班
105
Background: The use of metformin is associated with inhibition of oral squamous cell carcinoma proliferation and oral cancer transformation from oral premalignant lesion from reports of preclinical studies. However, due to the limitation of considering betel quid chewing habit as a major confounder, the chemopreventive effect of metformin on oral cancer remains controversial. Since the disorders of oral submucous fibrosis (OSF) are primarily appeared in betel quid chewers, investigation conducted on OSF patients would resolve the adjustment of betel quid chewing habits. The aim of this study is to investigate whether metformin is associated with reduced oral cancer risk in oral submucous fibrosis patients with type 2 diabetes and to further investigate whether there is a dose-response relationship. Methods: We conducted a population-based retrospective cohort study using Taiwan Health Insurance Research Database (NHIRD), Taiwan cancer registry and Death registry (DR) databases. The cohort consisted of newly-diagnosed oral submucous fibrosis patients with history of type 2 diabetes during 2010-2014. The 4 study groups were defined based on the usage of metformin within 6 months of before or OSF diagnosis: pre- & post-metformin users, pre-metformin only users, post-metformin only users and non-metformin users. The usage (with or without) of metformin may reflect possible selection bias at the diagnosis of OSF. To achieve better comparative groups, a matched study was conducted based on pre-metformin usage. Propensity scores were first calculated for patients with prior 6-month metformin usage, and then we matched one pre-metformin non-user to two pre-metformin users on the basis of the age, gender and propensity score. Kaplan-Meier estimates were used to compare oral cancer risk between four different groups. Multivariable Cox regression was used to estimate hazard ratios (HRs) of oral cancer between groups and investigate factors that associated with oral cancer risk. Results: There were 41 oral caners identified by Taiwan cancer registry from 1,031 OSF patients with type 2 diabetes. The results from Kaplan-Meier estimates showed no significantly difference of oral cancer risk among the four groups (log-rank p=0.2849). Cox regression analysis revealed that metformin used at post-diagnostic of OSF was associated with non-significant 0.42 times reduced oral cancer risk. In addition, OSF patients with type 2 diabetes who had cumulative DDD of metformin more than median cumulative DDD had non-significant 0.28 times (HR=0.28, 95% CI=0.019-4.229, p-value=0.3582) reduced oral cancer risk than patients who did not use metformin. Conclusion: Metformin may not have significant chemopreventive effect on oral cancer. In addition, there may not have significant dose-response relationship between metformin and oral cancer risk. Further epidemiological studies with larger sample size and randomized controlled trials are needed to investigate the association between use of metformin and oral cancer risk.

碩士
高雄醫學院
牙醫學研究所
85
Transforming growth factor beta (TGF-beta), a 25kd polypeptide of 112 amino acids , is a multi-functional growth factor that regulates the cell growth, differentiation and extracellular matrix formation (ECM) in both normal and transformed cells. This growth factor is secreted as a latent complex consisting of TGF-beta homodimer non-convalently associated with a dimer of latency-associated protein (LAP). Biologic activity is controlled by activation, when TGF-beta is dissociated from LAP by acid, base or protease.Increased activation of TGF-beta results in fibrogenic diseases such as liver cirrhosis, interstitial pulmonary fibrosis, glomerulo-nephritis, and skin fibrotic disease. These have been described by the fact that TGF-beta stimulates the deposition of collagen and other matrix components by fibroblasts, inhibits synthesis of collagenase, produces plasminogen activator inhibitor, enhances angiogenesis and is chemotactic for fibroblasts, monocytes, and macrophages. Oral submucous fibrosis (OSF) is characterized by an abnormal accumulation of collagen fibers in oral submucosa, and is also regarded as a precancerous lesion. Evidence of fibroblast proliferation, increased collagen formation and decreased collagenase activity in OSF has been described by many authors. Since the same biologic presentation of OSF in stimulating collagen deposition, fibroblast proliferation, inhibiting collagenase which result in accumulation of extracellular matrix, we suspect the TGF-beta may play a role in the OSF just like in other fibrogenic diseases.In this study, immunohistochemical staining was used to localize the existence of TGF-beta in clinical healthy oral mucosa and affected mucosa of OSF. The result showed that the incidence of TGF-beta expression over submucosal area in clinical healthy specimen(+7/14) is different from those in OSF(+23/29). Tissues from normal , inflammation and OSF also demostrate different patterns of TGF-beta expression. We concluded that there is significant difference (p<0.05) of TGF-beta expression between affected mucosa of OSF and clinical healthy oral mucosa.

碩士
高雄醫學大學
口腔衛生科學碩士在職專班
92
Background： The pathogenesis of oral submucous fibrosis(OSF) is unknown in medical reports. We try to find out those genes correlated with OSF. To explain the OSF disease was induced not only by betel by nut chewing but also by personal genes factors. Study objective： OSF is a chronic inflammatory and irreversed precancerous oral lesion. The etiologic factors in OSF are variant. We compared the genes and proteinsexpression profile between the normal oral tissues and OSF tissues by using cDNA micro-array and immunohistochemistry stainning to analysis the genetic change and protein identification respectively. Methods： Fifty four case of normal tissues from extracted mandibular third molar were included in this study as control group. Seventy two cases of clinical diagnosed OSF with evidence of histopathological proof and mouth open limitation with fibrosis band were included in this study as experimental group. Fresh tissues biopsy of oral mucosa were taken from the Department of Oral and Maxillofacial Surgery of Kaohsiung Military General Hospital. We used H-E stain method to differenciate the normal mucosa and OSF mucosa. The microarray analysis of genetic change and immunohistochemistry(IHC) stainning method were used to identify these parculiar proteins between the normal oral tissues and OSF tissues. After IHC stainning examination, we used Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) to identify the expression pattern of those genes. Results： The results revealed significant change in 1316 genes, with 18 genes being up-regulated, 10 genes being down-regulated and 6 genes being house keeping. The list of these genes including squamous cell carcinoma antigen I, P450 etc. they function as cell cycle, cell proliferation, cell apoptisis, protein metabolism, redox enzyme, transport factor and inflammation. From Independent-samples t Test analysis the Col I & Col III over OSF patient’s connective tissues had a statistically significant difference(P=0.000), Col III intensity 2.90±0.30 is higher than that of Col I 1.05±0.74. For same OSF patient, we compared the lesion side and normal side using the antibody Col I, Col III, PDK and P450. The results revealed a statistically significant difference(P=0.015) in P450 antibody among normal side connective tissues 2.40±0.52 and lesion side connective tissues 1.90±0.32. From the RT-PCR results, we compared the genes expression profile between the same OSF patient lesion side mucosa and normal side mucosa and normal tissues mucosa. We found the OSF lesion(Ratio＝1.95) side P450 genes expression 4 times lesser than normal tissues(Ratio＝7.49) and normal side(Ratio＝4.47) OSF genes expression 2 times lesser than for normal tissues. Conclusion： From micro-array analysis , IHC stainning, RT-PCR showed that results PDK、P450 had no significant difference in OSF and normal tissues mucosa, even they had high expression of micro-array genes analysis. These results provided a large amount of information regarding genes expression profiled which is associated with OSF. The no consistency expression in IHC stainning and RT-PCR may resulted from change in transcription and translation process genes expression profiled and proteins identification provided diagnostic, preventive and therapeutic utilization in future study of OSF.

碩士
中山醫學大學
牙醫學系碩士班
104
Oral submucous fibrosis （OSF） is a fibrosis disease because of chronic inflammatory stimulation oral epithelial atrophy and extracellular matrix (ECM) excessive deposition. OSF has been considered as a precancerous condition of oral mucosa, and according to the statistical research seen primarily in Southeast Asia. Betel nut chewing habits is inseparable with OSF pathogenesis, but the mechanisms of areca nut induced OSF are not well understood. The aim of this study was to investigate the epithelial-mesenchymal transition (EMT) transcription factor Snail expression in vitro, as known associated with many fibrotic diseases. We compare the Snail expression in primary buccal mucosal fibroblasts (BMFs) and primary oral submucous fibrosis-drived fibroblasts (OSFs), finding out Snail expression in OSFs is higher than that in BMFs, showing positive correlation between Snail and OSF. We used arecoline (major areca nut alkaloid) to explore whether expression of Snail could be changed dose in BMFs. We observed that the treatment of arecoline dose-dependently increased Snail expression transcript and protein levels in BMFs, while knockdown of Snail reversed these phenomena. Knockdown Snail expression inhibited migration and invasion in arecoline-stimulated BMFs and OSFs. These evidence suggest that knockdown Snail expression might be inhibited arecoline-stimulated myofibroblasts activation and proved Snail involved in the pathogenesis of OSF. This study hence attempts to provide insight EMT mechanism in the pathogenesis of OSF and new strategy for treatment of OSF patients.

碩士
國立臺灣大學
口腔生物科學研究所
95
ABSTRACT Oral submucous fibrosis (OSF) is a chronic debilitating disease and a pre-malignant condition of the oral cavity. It is characterized by a generalized submucous fibrosis. Epidemiological evidences strongly indicate the association of the areca quid (AQ) habit and OSF. It is regarded as a pre-cancerous condition. HDAC-2 (Histone deacetylase H2) has been evidenced to have over expression in some organs, such as lung, liver, and skin, but the underlying mechanisms remain to be clarified. In our study, we will discuss the pathogenesis between the severity of Oral submucous fibrosis (OSF) and the expression of HDAC-2 (Histone deacetylase H2). In the beginning, we collect 59 OSF specimens and 15 normal buccal mucosa that were examined the expression of HDAC-2 by immunohistochemistry (IHC). All patients received surgical excision of their precancerous at the Department of Oral and Maxillofacial Surgery, National Taiwan University Hospital, Taipei, Taiwan. The results of this study showed the HDAC-2 positive rate of OSF is significantly higher than that of normal oral mucosa (NOM) (P=0.0045).The incident rate of the OSF(ICR=96.6%）specimens, HDAC-2 is strongly expressed in the fibroblasts of connective tissue more than normal buccalmucosa（ICR=13.3%）specimens. In addition, the expression of HDAC-2 protein between OSF and NOM was quantified with the use of Western blotting and the results showed the same with IHC assay. Besides, SAHA (suberoylanilide hydroxamic acid) an HDAC-2 inhibitor was added to find the effect on OSF and NOM in a time- dependent manner by western blotting assay. And our studied indicated that after we added the SAHA for 12 hours this inhibitor is markedly reduced the expression of HDAC-2, especially in OSF (p<0.05). In other word, we can see the result suggests that In the OSF , HDAC-2 is strongly expressed in the fibroblasts of connective tissue protein more than normal buccal mucosa. And the result finds that HDAC-2 is higher expression and harder to be repressed in the OSF than in the NOM. Hence, the result suggests that over deposition of collagen maybe related to the over expression and hard to be repressed of HDAC-2 in the OSF.

碩士
國防醫學院
牙醫科學研究所
91
In order to identify the gene expression in the oral submucous fibrosis, we compare the gene expression profile to the normal tissues, oral submucous fibrosis tissue and oral cancer cell line by using cDNA microarray consisting 7597 known human genes. Comparison of expression profile between the normal tissues and oral submucous fibrosis tissue revealed significant change in 344 genes, with 316 genes being up-regulated and 28 genes being down-regulated. The list of these genes includes the classification of membrane proteins, hormones, cytoskeletons, transcription factors, enzymes, and cell cycle associated factors. The other comparison of the profiles between the normal tissues and SCC25 cancer cell line also revealed significant change in 720 genes, with 694 genes being up-regulated and 26 genes being down-regulated. By cross-compaing these biological informations, we found hOGG1 genes may be a marker of the carcinogenesis in OSF condition.Futher anaylsis of hOGG1 genes using the reverse transcription (RT) — PCR of original total RNA supported the reliability of our microarray analysis, More speciality, our study provide first evidence that hOGG1, c-ras, Staufen, IGFBP-3, Caveolin, Annexin V, cadherin 12, S-100 protein are up-regulated in the pathway of carcinogensis of oral submucous fibrosis. The results inform that the oxidative process may contribute the carcinogenesis of OSF. These findings provide a large body of information regarding gene expression profiles associated with oral submucous fibrosis pathogenesis, and also represent sourse of targets of oral submucous fibrosis prevention and therapeutics. Further validation of these genes using RT-PCR, Western blot analysis, and immunohistochemistry is undergoing.

碩士
中山醫學大學
生物醫學科學學系碩士班
103
In Taiwan, the behaviors of betel nut consumption and smoking cause several oral diseases including oral submucous fibrosis (OSF), which is considered as an oral precancerous condition. Current management of OSF includes stopping the areca quid chewing habit, medication and surgical intervention. However, there is no specific non-surgical drug in treatment of OSF being developed. Myofibroblasts, which are cells with positive expression of a-smooth muscle actin (a-SMA), are participated in fibrogenic process through collagen secretion. Resveratrol is an antioxidant polyphenol found in red wine and has been suggested to have heart protection effect by reducing cardiac fibrosis in the animal study. Here we would like to evaluate the therapeutic effect of resveratrol in treatment of OSF and determine the underlying molecular mechanisms. We first determined the effect of resveratrol in cell growth of two fibrotic buccal mucosal fibroblasts (fBMFs) and the IC50 is (131.3 ± 6.2) mM and (139.1 ± 19.9) mM, respectively. We further demonstrated that resveratrol could decrease the contraction capability of these two fBMFs with collagen contraction assay at the concentration of 100mM. By western blot, we found that Zinc finger E-box binding homeobox 1(ZEB1), a transcriptional factor in epithelial-mesenchymal transition process, was repressed by resveratrol simultaneously with inhibition of a-SMA and S100A4 expression in fBMFs. With quantitative polymerase chain reaction method, we also found that resveratrol inhibited ZEB1 expression at transcription level. With bisulfite pyrosequencing, we found that resveratrol treatment only slightly increased DNA methylation on two CpG islands within ZEB1 promoter region in fBMFs. We also discovered that treatment of resveratrol significantly increased miR-200c expression, the well-known microRNA with ZEB1-targeting activity. We further found that resveratrol treatment in fBMFs increased the expression of enhancer of zeste 2 polycomb repressive complex 2 subunit(EZH2) and trimethylated histone 3 at lysine 27 (H3K27me3), the substrate of EZH2. With chromatin immunoprecipitation analysis, the binding of H3K27me3 on ZEB1 promoter in fBMFs was also enhanced by resveratrol. Finally, knockdown of EZH2 in fBMFs increased the expression of ZEB1. In conclusion, resveratrol displays an anti-OSF activity through epigenetic control of ZEB1 expression.

博士
高雄醫學大學
牙醫學系博士班
106
Oral submucous fibrosis (OSF) is an oral potentially malignant disorder and areca nut chewing is the main etiological factor of this lesion in regions with high prevalence of areca nut chewing habits. However, the molecular mechanism underlying OSF remains unknown, partly due to the lack of an appropriate animal model. The present study aims to establish and characterize two animal models of areca nut extract (ANE)-induced (1) skin fibrosis and (2) oral submucous fibrosis. In skin fibrosis model, 24 mice were equally divided into 4 groups (control, bleomycin, ANE10 and ANE20 groups) and subcutaneous (SC) injection every two days. In OSF model, 18 mice were equally divided into 3 groups (control, bleomycin and ANE 20 mg/ml groups) and subcutaneous injection was applied once every week. Six mice from each of skin group were killed via CO2 inhalation after the treatments had been administered for 3, 7, 14, and 30 days, whilst all the animals of oral groups were sacrificed after 14 and 30 days. Then, the fibrotic formation of the tissue was evaluated by histological analyses; the expression levels of the fibrotic marker proteins were assessed by immunohistochemical staining and immunoblotting. In ANE-induced skin fibrosis, ANE administration significantly increased dermal thickness and collagen deposition when compared with the control group. Moreover, ANE increased the expression of the fibrotic marker genes alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) respectively in the skin and submucous lesions. In ANE-induced OSF, ANE administration significantly increased lamina propria thickness and collagen deposition. The SC injection of ANE successfully induced skin fibrosis, exhibiting characteristics similar to those of OSF. Moreover, these two models may facilitate future studies of the possible therapeutic method for OSF. Therefore, we tried to use two treatments: low power laser irradiation (LPLI) and forskolin (an intracellular cAMP activator). To the result showed that both treatments have therapeutic effects. However, we still need to further investigate the mechanisms of the treatments for OSF, which have indelible benefits for development of OSF clinical interventions. It is expected that the established OSF animal models are valuable to verify a potential treatment for OSF patients in the future.

碩士
國立臺灣大學
牙醫學研究所
82
In this study, we used immunohistochemical method to assess the expression of p21 ras, p53 and proliferating cell nuclear an- tigen (PCNA) in 50 cases of oral submucous fibrosis (ＯＳ Ｆ), 10 cases of normal oral mucosa, 10 cases of leukoplakia, 5 cases of epithelial dysplasia, and 5 cases of verrucous carcinoma. By statistic analysis, we further correlated the expression of p21 ras, p53 and PCNA in ＯＳＦ with betel nut chewing, cigarette smoking, alcohol drinking as well as the depth of fibrosis and the degree of inflammation in subepithelial connective tissue. By immunohistochemistry, the positive staining of p21 ras was most often found in the cytoplasm and nuclei of epithelial cells of ＯＳＦ oral mucosa. The pattern of p21 ras positive staining of ＯＳＦwas similar to that of normal oral mucosa. The positive staining of p53 was most frequently discovered in the nuclei of basal and suprabasal epithelial cells of ＯＳＦ oral mucosa. The pattern of p53 positive staining of ＯＳＦ was similar to those of leukoplakia and epithelial dysplasia. The positive staining of PCNA was most commonly noted in the nuclei of basal, suprabasal and lower spinous epithelial cells. The pattern of PCNA positive staining of ＯＳＦ was very close to those of leukoplakia, epith- elial dysplasia, and verrucous carcinoma. The expression of p53 in the whole layer of epithelial cells of ＯＳＦ oral mucosa was positively and significantly correlated to the duration of chew- ing betel nuts (P<0.05). There was a significant negative corre- lation between the expression of PCNA in the whole layer of epi- thelial cells of ＯＳＦ oral mucosa and the total number of ciga- rettes smoked by the ＯＳＦ patients (P<0.05). We concluded thatＯＳＦ was undoubtful an oral precancerous lesion, because the p53 and PCNA positive staining patterns of ＯＳＦ were similar to those of oral leukoplakia and epithelial dysplasia.

碩士
中山醫學院
營養科學研究所
88
Abstract Oral submucous fibrosis (OSF), an obscure oral pathosis, is characterized by an abnormal accumulation of oral submucous collagen fibers, epithelium atrophy, and the resultant limitation of mouth opening. Studies indicated that OSF was one of precarcinogenic lesions and mortality of oral cancer is the fifth cause of cancer-related death among men. The reasons for the occurrence of OSF and oral cancer were still unknown. Some studies showed that it is closely related to nutritional deficiency, but it is limited to the related evidence. The purpose of this study is to understand the life style, diet behavior, nutritional assessment and immune function of patients with OSF and oral cancer, and the data colleted could be served as a reference on clinical therapy. Subjects were recruited from Taichung and Changhua. To understand the life style, diet behavior and daily intake of patients with OSF and oral cancer. The subjects got to complete a life a style questionnaire, 24-h diet recall, and the blood samples were collected to examine the nutritional status and immune function. The results show that 96.4% of OSF and oral cancer patients had the habit of chewing betel quid. And, 92.8% of them favored smoking and 65% of them favored drinking. The intake of energy, vitamin E, B2, B6, C, niacin, calcium, and iron for all patients were all lower than daily recommended nutrient allowance (RDNA). The nutritional status showed that the content of vitamin E, B1, selenium and zinc were lower in all patients and vitamin B6 was decreased with the severity of disease. The evaluation of the immune showed that, the relative percentage of T-lymphocyte and the ratio of CD4 and CD8 were also gradually decreased with the severity of disease. In conclusion, patients with OSF and oral cancer had poor diet intake, malnutrition and depressed immune-response sensitivity, etc. Correct diet advice and nutrition supplement could be greatly helpful to prevention and clinical therapy of OSF and oral cancer. Key words: OSF, oral cancer, nutrition, immune, diet behavior

Magister Scientiae Dentium - MSc(Dent)
Betel nut/quid chewing is a habit that is commonly practiced in the Indian subcontinent. This age-old social habit is still practiced by Indians in Durban, Kwazulu Natal (South Africa). The betel nut/quid is prepared in a variety of ways. The quid may be prepared with or without tobacco. This habit is said to be associated with the development of premalignant lesions,namely, Oral Submucous Fibrosis (OSF) which increases the susceptibility for malignancy of the oral mucosa and the foregut. The aim of this study was to investigate the prevalence of betel nut/quid chewing (with or without tobacco), the associated habits (smoking and alcohol consumption) and awareness of the harmful effects of the chewing habit among Indians in Durban, KwaZulu-Natal.A cross-sectional study design was chosen utilising a self-administered questionnaire and semi-structured interviews to collect data. Consenting participants were requested to complete a self-administered, structured questionnaire. The study population included any person in the Durban area who chewed betel nut/quid/tobacco. Only persons willingly and who consented to be part of the study, were included. The sample size was based on convenience. People were approached at the pan shops, leisure markets, traditional functions and at the dental practice the researcher operated at. A total of 101 respondents were interviewed.A significantly higher proportion of females chewed betel nut/quid from the total of the respondents. The results showed that the habit is increasingly practiced in the younger age group (20-39 years). There was evidence to show that the chewing habit is used more by the employed than the unemployed (p=0.055). Of the sample population, 78% were born in South Africa and the rest were immigrants from Pakistan, India and Dubai. All respondents from the migrant community were males. The most important reasons for chewing betel nut were for enjoyment and at special functions. More than two third indicated family members (aunts,uncles and cousins) influence as a reason for chewing, in comparison to influences by parents or grandparents. The study also indicated that parents were far more likely to influence betel nut chewing if grandparents did so (p-value= 0.000). In addition, the study revealed that family members (aunts, uncles and cousins) were far more likely to influence betel nut chewing if parents did so (p=0.000).The most popular ingredients chewed were betel nut, betel leaf, lime and pan masala and the most popular combinations were betel nut/lime/betel leaf quid preparation, betel nut alone,betel nut/betel leaf/lime/tobacco/pan masala and betel nut/betel leaf/lime/pan masala. Two thirds of the respondents do not know that betel nut chewing is harmful to their health, thus indicating a lack of awareness on the risks associated with the chewing habit, and the majority have not attempted to give up the habit. Most of the respondents retained their chewing habits after being informed about the risks. A little more than half the study population reported neither smoking nor drinking.The present study found that betel nut/quid chewing habits continue to be enjoyed by many people and most are unaware of the hazardous effects of the habit. More younger people are using the habit as compared to previous studies. This is probably because it is an affordable and easily accessible habit. It is recommended that aggressive awareness programmes on the harmful effects of betel nut/quid chewing be developed, similar to that for smoking cessation.Government health warnings need to be instituted, for example, by having written warnings on packagings. Taxes need to be imposed on the betel nut and condiments thereby reducing access to most people. Age restrictions need to be imposed on purchasing of the betel nut/quid thus making access difficult for the children.

碩士
高雄醫學大學
牙醫學研究所
89
Oral cancer has become a series health issue in Taiwan, and it is believed that oral submucous fibrosis(OSF) and leukoplakia are precancerous lesions. Therefore to focus on the etiology of these oral mucosal lesions is very important for the prevention and treatment of oral cancer. Oral precancerous mucosal lesions have a close link to the immune systems and cytokines have played an important role on how to regulate the immune systems. It is important that human peripheral blood mononuclear cells could produce cytokines and it has become a guideline of how to evaluate the cellular immunity of these patients. In our study, we cultured the peripheral mononuclear cells of the patients with OSF and leukoplakia and detected the mRNA expression of TNF-α、TGF-β and IFN-γ by RT-PCR method. Our result showed that the TNF-α and IFN-γ mRNA expression is higher in OSF patients and TGF-β may have different expressions because of different stages of OSF. TNF-α and IFN-γ mRNA expression is higher in leukoplakia patients but TGF-β mRNA expression is lower in patients with leukoplakia than in normal patients.