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1
Burns, Jason Lee. "Growth control by insulin-like growth factor II." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270285.
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Mörth, Corinna. "Consequences of postnatal insulin-like growth factor II overexpression in insulin-like growth factor I deficient mice." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-46307.
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Horn, Henrik von. "Regulation of insulin-like growth factor-II in human liver /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-880-0/.
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Zhang, Qimin. "Insulin-like growth factor II : cellular effects through different receptors /." Stockholm, 1998. http://diss.kib.ki.se/1998/91-628-2754-5.
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Gadd, Stephanie Clare. "Insulin-like growth factor II in preovulatory follicles and ovarian cysts." Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296517.
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Ohlsen, Susan M. "Cloning and characterization of ovine insulin, insulin-like growth factor-I and -II genes." Diss., This resource online, 1993. http://scholar.lib.vt.edu/theses/available/etd-06062008-165729/.
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Gustafsson, Sara. "The insulin-like growth factor system - effects of circulating proteases /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-436-8/.
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Olausson, Hanna. "Nutritional status before and during pregnancy in relation to the maternal insulin-like growth factor-system and health related variables in the offspring : studies in women, guinea pigs and rats /." Linköping : Univ, 2004. http://www.bibl.liu.se/liupubl/disp/disp2004/med860s.pdf.
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Elliss, Carolyn. "Studies of the role of IGF-II during mouse development." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291034.
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Makins, Richard John. "Insulin-like growth factor-II in colitis and colitis-associated colorectal cancer." Thesis, Queen Mary, University of London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419706.
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Lee, Adrian. "Analysis of insulin-like growth factor-II in human breast cancer cells." Thesis, University of Surrey, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335638.
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Mukherjee, Sudipto. "Retinal pigment epithelial cells and the insulin-like growth factor system in proliferative vitreoretinopathy." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/mukherjee.pdf.
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Petrik, James J. "The role of insulin-like growth factor-II as a pancreatic mitogen and survival factor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0034/NQ58157.pdf.
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Diehl, Daniela. "Bedeutung von Insulin-like growth factor II (IGF-II) und IGF-Bindungsprotein-4 in der Kolonkarzinogenese In-vitro- und In-vivo-Studien /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973069422.
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Hedman, Christina A. "Insulin and IGF-I in type 1 diabetes /." Linköping : Univ, 2005. http://www.bibl.liu.se/liupubl/disp/disp2005/med915s.pdf.
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Robinson, Rose Marie. "Microphysiometry Studies of Rapid Binding of Insulin-Like Growth Factor I by Parental and Transfected Mammary Epithelial Cell Lines." Thesis, Virginia Tech, 1998. http://hdl.handle.net/10919/35363.
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Breast cancer is a leading cause of cancer death of women in the U.S. today. Members of the family of insulin-like growth factors (IGFs) are proposed to play a major role in the development and subsequent uncontrolled proliferation of breast cancer cells. Insulin-like growth factor-I (IGF-I) is known to be a potent mitogen for mammary epithelial cells. IGF-I acts by binding to cell surface receptors, thereby stimulating a cascade of events leading to cell division. In the interest of interrupting the effect of IGF-I on cancerous mammary epithelial cells, an understanding of how IGF-I behaves in the presence of other extracellular components is needed. This study examines the IGF-I response of SV40-IGF-I, an immortalized bovine mammary epithelial cell line which secretes IGF-I constitutively.
The microphysiometer allows real-time sampling of cellular activity by measuring the excretion of protons from a sample of cells stimulated by IGF-I binding. The contributions of other factors in enhancing or suppressing stimulation can be compared by examining the pH response of cells exposed to IGF-I in the presence of these factors. We present data showing the stimulatory effect of IGF-I in a dose dependent manner on the SV40-IGF-I cell line.
In addition, we compare IGF-I stimulation with stimulation by long R3IGF-I, a substituted analogue of IGF-I having a reduced binding affinity for the IGF binding proteins. We examine the effect of insulin-like binding protein-3 (IGFBP-3) both in the presence and absence of IGF-I, finding no IGF-I independent effect in the rapid binding experiment and no effect on stimulation of IGFBP-3 pre-incubated cells by subsequent IGF-I challenge. This is of particular interest due to recent work demonstrating an IGF-independent IGFBP-3 response in a number of cell lines. Binding studies to correlate with the rapid binding stimulation show binding of the IGFBP-3 molecule with high affinity to a small number of surface receptors on the SV40-IGF-I cell.
Analysis of the extracellular environment and the components contributing to the binding of IGF-I to the cell membrane receptor will provide information for the development of interventions to slow or interrupt the process of IGF-I binding and therefore cancer growth. Optimization of the Cytosensor(r) Microphysiometer System for the (transfected) SV40-IGF-I and the (parental) MAC-T cell lines was achieved to continue comparison studies of autocrine and paracrine stimulation of bovine mammary epithelial cells by IGF-I.
This work was supported by the Whitaker Foundation Biomedical Engineering Grant.Master of Science
17
Craven, Cyril John. "The mechanism of maturation of insulin-like growth factor-1." Thesis, Queensland University of Technology, 1999. https://eprints.qut.edu.au/37030/7/37030_Digitised%20Thesis.pdf.
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This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which
would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l-
3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike
activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however,
detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed
unlikely to participate in the formation of des(1-3)IGF-I.
In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely
that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of
an in vivo role for des(1-3)IGF-I, as reported by others.
The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with
a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the
presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu.
As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation
with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal
GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I.
The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial
polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to
des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would
argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS.
No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this
approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum.
In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to
"mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1-
3)IGF-I as a transient product which is bound to the receptor and internalised.
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Giannoukakis, Nick. "The genetic and epigenetic regulation of insulin-like growth factor II gene expression in humans." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0014/NQ36977.pdf.
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Li, Juan. "Regulation of insulin-like growth factor II gene expression in the late gestation fetal sheep." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336613.
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Kiefer, Julie Christine. "Analysis of myogenic regulatory factors and insulin-like growth factors in early somite myogenesis /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9227.
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Gaunt, Thomas Richard. "Variation in the gene for insulin-like growth factor II and its relationship with anthropometric traits." Thesis, University of Southampton, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249600.
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Voute, Lance Caesar. "Investigation of the role of insulin-like growth factor-I and -II and insulin in the pathogenesis of equine osteochondrosis." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608807.
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Clairmont, Kevin B. "The Structure, Function, and Regulation of Insulin-like Growth factor II/Mannose 6-phosphate Receptor Forms: a Thesis." eScholarship@UMMS, 1990. https://escholarship.umassmed.edu/gsbs_diss/44.
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In mammals a single receptor protein binds both insulin-like growth factor II (IGF-II) and mannose 6-phosphate (Man 6-P) containing ligands, most notably lysosomal enzymes. However, in chick embryo fibroblasts IGF-II binds predominantly to a type 1 IGF receptor, and no IGF-II/Man 6-P receptor has been identified in this species. In order to determine if chickens possess an IGF-II/Man 6-P receptor, an affinity resin (pentamannosyl 6-phosphate (PMP) Sepharose) was used to purify receptors from chicken membrane extracts by their ability to bind mannose 6-phosphate. Then 125I-IGF-II was used to evaluate their ability to bind IGF-II. These experiments demonstrate that nonmammalian Man 6-P receptors lack the ability to bind IGF-II, suggesting that the ability to bind IGF-II has been gained recently in evolution by the mammalian Man 6-P receptor.
The second area of study involves the serum form of the IGF-II/Man 6-P receptor. This receptor had been detected in the serum of a number of mammalian species, yet its structure, function, regulation, and origin were unknown. Initial studies, done with Dr. R. G. MacDonald, showed that the serum receptor is truncated such that the C-terminal cytoplasmic domain of the cellular receptor is removed. These studies also demonstrate a regulation of serum receptor levels with age, similar to that seen for the cellular receptor, and that the serum form of the receptor existed in several forms which appeared intact under nonreducing conditions, but as multiple proteolytic products upon reduction. Finally, these studies demonstrated that both the cellular and serum IGF-II/Man 6-P receptors are capable of binding IGF-II and Man 6-P simultaneously.
In studies on the serum form of the IGF-II/Man 6-P receptor that I have conducted independently, the regulation of the serum IGF-II/Man 6-P and transferrin receptors by insulin has been demonstrated. In these studies, insulin injected into rats subcutaneously resulted in a time and dose dependent increase in serum receptor levels. Finally, to investigate the relationship of the serum IGF- II/Man 6-P receptor to the cellular form of the receptor, pulse chase experiments were performed. These experiments demonstrate that the soluble (serum form released into the medium) receptor is a major degradation product of the cellular receptor. Furthermore, the lack of detectable amounts of the lower Mr soluble receptor intracellularly and the parallel relationship of cell surface and soluble receptor suggest that the proteolysis is occurring from the cell surface. Finally, a number of experiments suggest that the degradation rate depends upon the conformation state of the receptor: binding of IGF-II or Man 6-P makes the receptor more susceptible to proteolysis while the presence of lysosomal enzymes prevents receptor proteolysis.
In summary, the serum form of the IGF-II receptor is a proteolytic product of the cellular form of the receptor. The rate of release depends upon the number of receptors at the cell surface and the binding state of the receptor. In circulation, the receptor retains the ability to bind both types of ligands, it thus may serve as an IGF binding protein and/or a lysosomal enzyme binding protein. These results suggest a model whereby the cellular receptor is proteolytically cleaved by a plasma membrane protease to produce a short membrane anchored fragment and the serum receptor. In vivo this pathway serves as the major degradative pathway of the IGF-II/Man 6-P receptor, with the serum form being cleared from circulation by further degradation and reuptake.
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Sun, Fang-Lin. "Overexpression of the mouse insulin-like growth factor II gene : a transgenic mouse model for human Beckwith-Wiedemann syndrome." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627341.
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Coletta, Rocio Riatto Della. "Análise das repetições CA do gene IGF1, VNTR do gene da insulina e região promotora P4 do gene IGF2 em indivíduos nascidos pequenos para idade gestacional." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-29042008-144128/.
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Introdução: Polimorfismos na região promotora dos genes da insulina, IGF2 e IGF1 podem estar relacionados a uma diminuição da expressão desses genes na vida fetal que, por sua vez, pode causar restrição do crescimento intra-uterino e maior risco de hipospádia. Na vida pós-natal, perda completa ou parcial da expressão desses genes pode resultar em ausência de recuperação estatural e menores concentrações séricas de IGF1 na criança, além de um maior risco de diabetes melito tipo 2 e síndrome de resistência à insulina no adulto. Objetivos: Analisar em crianças nascidas pequenas para idade gestacional (PIG) com ou sem recuperação estatural (RE): 1) a freqüência alélica e genotípica dos polimorfismos VNTR-INS e das repetições CA do gene IGF1; 2) a região promotora P4 do gene IGF2; 3) a influência do VNTR INS e das repetições CA do gene IGF1 na sensibilidade à insulina e nas concentrações séricas de IGF1, respectivamente. Pacientes: Foram estudados 142 indivíduos nascidos PIG com (n= 66) e sem recuperação (n= 76) estatural selecionados de três diferentes centros (HC-FMUSP, Santa Casa de São Paulo e HC-UFPR) e um grupo controle constituído de 297 indivíduos nascidos adequados para idade gestacional (AIG). Métodos: Extração de DNA genômico; amplificação por PCR das regiões contendo os polimorfismos VNTR INS e repetições CA do IGF1 e da região promotora P4; digestão por enzima de restrição; software Genescan; seqüenciamento automático; avaliação bioquímica e hormonal da glicemia, insulina e IGF1, extração de RNA, PCR em tempo real e análise estatística com SPSS 13.0 (Statistical Package fo Social Sciences). Resultados: A média do Z-altura, Z-IMC (índice de massa corpórea), Z-altura paterno e ZEA (estatura alvo) foram maiores nas crianças PIG que tiveram recuperação estatural, com o Z-PC (perímetro cefálico) maior nas crianças sem recuperação estatural. O Z-IGF1 sérico foi significantemente mais elevado em crianças que apresentaram RE (p<0,05). A distribuição e genotipica das repetições CA do gene IGF1 e do VNTR INS foi semelhante estatisticamente entre os grupos AIG e PIG, e entre os PIG com e sem RE; não foi observada associação entre esse polimorfismo e as variáveis clínicas e laboratoriais do estudo. O estudo da região promotora P4 do gene IGF2 identificou um novo polimorfismo de 9-12 repetições C na posição -1982, antes do sítio de início de transcrição do exon 2, e este apresentou distribuição semelhante entre os grupos PIG e AIG. Foi identificada também uma troca C/T em heterozigose no nono nucleotídeo do alelo 11C em quatro crianças nascidas PIG. Contudo, a quantificação da expressão do gene IGF2 em duas dessas crianças não demonstrou perda da expressão desse gene. Conclusões: Não observamos influência dos polimorfismos acima descritos no crescimento pré e pós-natal, na presença de resistência à insulina, nem em concentrações séricas de IGF1 dos indivíduos nascidos PIG. Identificamos uma nova variante na região promotora P4 do gene IGF2, contudo estudos preliminares não demonstraram influência desse polimorfismo sobre o crescimento intra-uterino.Introduction: Polymorphisms in the promoter region of insulin (INS), IGF2 and IGF1 genes may decrease their expression during fetal life and afterward could be related to intra-uterine fetal growth retardation and greater risk of hypospadia development. In post-natal life, decreased expression of these genes can result in lack of stature recovery and in lower IGF1 serum levels in children, as well as in higher risk for type 2 diabetes mellitus and metabolic syndrome in adults. Objectives: The aims of the present study were: (1) to analyze the allelic and the genotypic frequency of the insulin (INS) gene variable number of tandem repeats (VNTR) and the IGF1 gene CA repeats; (2) to analyze the P4 promoter region of IGF2 gene (3) to test the contribution of INS VNTR, IGF1 gene CA repeats on insulin sensitivity and IGF1 serum levels in children born SGA with and without catch up, respectively. Patients: We studied 142 individuals born SGA with catch up (n = 66) and without catch up (n = 76) selected from three different centers (HCFMUSP, Santa Casa de Sao Paulo and HC-UFPR). The control group consisted of 297 children born appropriate for gestational age (AGA). Methods: Extraction of genomic DNA, PCR-amplification of the VNTR of insulin gene, CA repeats of IGF1 and IGF2 gene P4 promoter region; restriction analysis; Genescan software; automatic sequencing. Blood measurements of serum level of glucose, insulin and IGF1. Statistical analysis (Statistical Package for Social Sciences software). Results: Regarding birth parameters, the average of Z-height, Z-BMI (body mass index) and Z-height paternal and Z- EA (target height) were higher in children born SGA who had catch up. Interestingly, we observed that the Z-PC was higher in children born SGA without catch up. In addition, the Z-IGF1 serum levels were significantly higher in children who had catch up (p <0.05). The molecular analysis of IGF1 gene CA repeats and of INS gene VNTR locus did not show a statistically significant difference in the allelic and genotypic distribution of these polymorphisms between adequate for gestational age (AGA) and SGA groups nor between SGA with and without catch up. Similarly, we have not found an association of these polymorphisms with clinical or laboratory variables of this study. A novel polymorphism in the P4 promoter region of the IGF2 gene was identified. It was characterized by cytosine repeats (9-12) at position -1982 before transcription initiation site of exon 2 of IGF2 gene. Yet, we have identified a heterozygous substitution of cytosine for thymine at the nucleotide position 9 in the allele 11C in four children born SGA. This change was also absent in the control population. Quantization of IGF2 gene expression in two of these children did show loss of expression of this gene in patients carrying the variant 9C/T. Conclusions: We have not observed an association of the above described polymorphisms with pre and post natal growth, or with the occurrence of insulin resistance in individuals born SGA. IGF-1 levels did not seem to be associated with the polymorphisms either. A new variant in the P4 promoter region of IGF2 gene was identified, however preliminary studies showed no influence on intra-uterine growth.
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Montenegro, Luciana Ribeiro. "Estudo in vitro da sensibilidade ao IGF-1 de fibroblastos de crianças nascidas pequenas para a idade gestacional sem recuperação estatural pós-natal." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-08092009-132008/.
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Introdução: Crianças nascidas pequenas para a idade gestacional (PIG) apresentam maior risco de permanecerem com baixa estatura na vida adulta. Os fatores de crescimento insulino-símile tipo 1 e 2 (IGF-1 e IGF-2) são os principais fatores endócrinos determinantes do crescimento fetal. A maioria das ações conhecidas do IGF-1 e 2 é mediada via um receptor tirosina quinase, conhecido como IGF-1R. Recentemente, a insensibilidade ao IGF-1 foi identificada como uma das causas de retardo de crescimento em crianças nascidas PIG que não apresentaram recuperação espontânea do crescimento na vida pós-natal. Crianças afetadas apresentavam níveis elevados de IGF-1, IGFBP-3 além de microcefalia. O papel de defeitos pósreceptor na sinalização do IGF-1 como causa de retardo de crescimento pré e pós-natal ainda não foi investigado. Objetivo: Analisar in vitro a ação do IGF-1 em fibroblastos de crianças nascidas PIG. Material e métodos: Desenvolvemos cultura de fibroblastos de 2 controles (C1 e C2) e de 4 pacientes nascidos PIG (SGA1, SGA2, SGA3 e SGA4) com suspeita de insensibilidade ao IGF-1 por ausência de recuperação do crescimento na vida pós natal, resposta insatisfatória ao tratamento com hGH apesar de níveis normais/elevados de IGF-1. Foi confirmado do ponto de vista molecular que um dos pacientes (SGA1) apresenta Síndrome de Sílver- Russell com perda da metilação do alelo paterno da região ICR1 (imprinting center region 1) importante para a expressão do IGF-2. Defeitos no gene do IGF1 e IGF1R foram afastados por sequenciamento direto. As ações do IGF- 1 foram determinadas por ensaios de proliferação, análise da produção de IGFPB-3 em meio de cultura e estudos de fosforilação de proteínas da via de sinalização do IGF-1 em fibroblastos (AKT e ERK). Resultados: As linhagens SGA1, SGA2 e SGA3 proliferaram respectivamente 31%, 60% e 78% a menos sob estímulo de IGF-1 em relação ás linhagens controles. Já a linhagem SGA4 apresentou comportamento semelhante ás linhagens controles. No estudo da expressão do RNAm do IGF1R por PCR em tempo real, não foi observada diferença significativa na expressão do IGF1R nas diversas linhagens PIG em relação aos controles, assim como o conteúdo total da proteína IGF-1R. Em relação á ativação da via MAPK, todas as linhagens dos pacientes PIGs apresentaram menor fosforilação ERK1/2 basal e após estímulo com IGF-1, quando comparadas com as linhagens controles (p < 0.001) apesar do conteúdo total de ERK1/2 ser semelhante. Já em relação a ativação da via PI3K, as linhagens SGA1, SGA2, SGA3 e SGA4 não diferiram significantemente em relação aos fibroblastos controles quanto à ativação de AKT pelo IGF-1. O conteúdo total de AKT também foi semelhante em todas as linhagens estudadas. O estudo da expressão de IGFBP3 mostrou um aumento da expressão deste peptídeo na linhagem de fibroblastos do paciente SGA1 (14X). O conteúdo de IGFBP-3 intracelular não sofreu alteração, porém comprovamos que a linhagem SGA1 secretava 2x mais IGFBP-3 para o meio de cultura. Apesar de apresentarem estrutura, expressão e conteúdo de IGF1R normais, essas mesmas 3 linhagens celulares que apresentaram menor proliferação também apresentaram diminuição na fosforilação de ERK após tratamento com IGF-1. Mesmo sob o estímulo com desIGF-1 (um análogo do IGF-1 com baixa afinidade por IGFBPs mas que preserva sua capacidade de ativar o receptor IGF-1R) a ativação de ERK e a proliferação celular se manteve abaixo dos das linhagens controles. O estudo do conteúdo total de GRB10 foi semelhante em todas as linhagens celulares. Conclusão: Três dos 4 pacientes PIG estudados apresentaram insensibilidade pós-receptor ao IGF-1. A linhagem celular SGA1, obtida de um paciente com hipometilação do ICR1 11p15 causando SSR, demonstramos um aumento da expressão e secreção de IGFBP-3, o qual não se mostrou responsável por inibir a ação do IGF-1 nestes fibroblastos. Novos estudos devem ser desenvolvidos para identificar o defeito molecular responsável pela insensibilidade ao IGF-1 a nível pósreceptor observada nestes pacientes.Introduction: Children born small for gestational age (SGA) have a higher risk of staying with short stature in adulthood. The insulin-like growth factors (IGF-1 and IGF-2) are the main endocrine factor determining fetal growth. Most of the known actions of IGFs are mediated by IGF-1R, a tyrosine kinase receptor. Recently, the IGF-1 insensitivity was identified causing growth retardation in children born SGA who who did not present spontaneous catch-up growth in postnatal life. Affected children had elevated IGF-1 and IGFBP-3 levels in addition to microcephaly. The role of post receptor defects in IGF-1 signaling on the deficit of growth is still unclear. Objective: To assess IGF-1 action and signaling in vitro in fibroblasts from SGA children. Methods: Fibroblasts cell cultures were developed from 2 controls (C1 and C2) and 4 patients with pre- and post-natal growth retardation (SGA1, SGA2, SGA3 and SGA4). IGF-1 insensitivity was demonstrated by severe pre and postnatal growth impairment without any evident cause, IGF1 SDS > 0 and poor growth response during high doses of hGH treatment. Three SGA patients presented microcephaly. Defects in the gene of the IGF1 and IGF1R were excluded by direct sequencing. One patient (SGA1) presents the Silver- Russell syndrome (SRS) with loss of methylation of the paternal allele in the ICR1 (imprinting center region 1) chromosome 11p15, important for IGF-2 expression. IGF-1 action was assessed by cell proliferation by colorimetric assay. IGF-1 signaling was assessed by AKT and ERK phosphorylation after IGF-1 stimulation through SDS-PAGE of intracellular extract followed by immunoblotting with specific antibodies. The expression of IGF1R and IGFBP3 gene was determined by Real-time quantitative PCR and the levels of the IGF-1R and IGBP-3 protein by direct immunoblotting. Results: The SGA1, SGA2 and SGA3 cell lines proliferated 31%, 60% and 78% less under IGF-1 stimulation in comparison of controls fibroblasts, respectively. The expression of IGF1R mRNA and the level of total amount of IGF-1R protein were similar in all SGA and control cell lines. Despite normal IGF-1R structure and quantity, the same 3 SGA cell lines that presented low proliferation response also had 50 to 85% lower ERK phosphorylation after IGF-1 treatment (p <0.001), although the similar total content of ERK1/2. In relation to PI3K pathway activation, all SGA cell cultures presented normal AKT phosphorilation. Fibroblasts from the SGA1 patient presented a 14x increase in IGFBP3 mRNA and 2x more IGFBP-3 secretion to culture serum medium. Treatment with desIGF-1, an IGF-1 analogue with low affinity for IGFBPs although retains its ability to activate the IGF-1R, did not recover cell proliferation or ERK phosphorylation. All cell lines presented similar amount of GRB10 protein Conclusion: Three of 4 SGA patients showed evidence of post-receptor IGF-1 insensitivity. The cell line SGA1, obtained from a SRS patient with ICR1 hypomethylation, showed increased expression and secretion of IGFBP-3, which was not directly responsible for inhibition in IGF- 1 action. Further studies should be developed to identify the molecular cause of IGF-1 post-receptor insensitivity observed in our patients.
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Blanchard, Frédéric. "Contribution a l'etude de la cytokine lif et de ses recepteurs (doctorat : immunologie-biologie)." Nantes, 1998. http://www.theses.fr/1998NANT16VS.
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Alazzabi, Mufida. "Insulin-like growth factor-II (IGF2) gene of zebrafish and its use as a biogenetic marker for the assay of epigenetic toxin exposure." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26561.
Full textAbstract:
The purpose of this research was to determine whether expression analysis of the IGF2 gene in zebrafish, a gene whose transcription is known to be regulated by DNA methylation in mammals, can be used as a marker or indicator of DNA methylation due to toxin exposure in fish embryos. We examined the expression of IGF2, IGF1, and IGFBP-1 in zebrafish embryos treated with sodium arsenite (thought to inhibit DNA methylation), nickel chloride (thought to cause DNA hypermethylation), trichostatin A (a histone deacetylase inhibitor), 5-azaC (thought to cause methyltransferase inhibition), cadmium chloride (thought to cause DNA hypermethylation) and mercury chloride (unknown). (Abstract shortened by UMI.)
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Milosevic, Barbara. "The development of a non-lytic insect-cell expression system for the production of fusion proteins of insulin-like growth factor II and stromal-cell derived factor 1 /." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31273.
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Insulin-like growth factors (IGFs) and stromal cell derived factor 1 (SDF-1) have been suggested as being potential therapeutic agents in HIV infected patients. The cDNA coding for a recombinant fusion protein of IGF 1I and SDF-1 was synthesized by PCR and inserted into a commercial non-lytic insect cell expression system. The successful production of the recombinant protein in the culture medium of insect cells transfected with the recombinant plasmid was monitored using HPLC. Purified protein was characterized by following 3H-thymidine incorporation into bovine fetal liver cells for the identification of IGF II and using Western blots and enzyme-linked immunosorbent assays for the identification of SDF-1.A modified IGF II-SDF 1 chimera, which included a N-glycosylation site, was also successfully produced by insect cells. Mass spectrometry analysis indicated that the glycosylated and non-glycosylated proteins had molecular masses of 17.6 kDa and 14.5 kDa, respectively. The recovery of the glycosylated protein was higher than that obtained with the non-glycosylated form after purification by heparin affinity chromatography, a property that could be an advantage for the large scale production of the protein. Finally, three additional cDNAs were successfully generated and cloned into insect cell expression vectors. Two of them encoded the glycosylated-and non glycosylated forms of the chimera incorporating a Kozak sequence to improve translation efficiency. The third cDNA encoded a chimera of IGF II and a soluble form of the HIV-binding protein CD4, a potential inhibitor of HIV infection.
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Xu, Yongqin. "Studies on parental genomic imprinting of insulin-like growth factor-II/mannose 6-phosphate receptor gene in humans, phenomenon, mechanism, and relevance to disease." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0019/NQ44633.pdf.
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Mainville, Gisele Nadia. "The Prognostic Significance of Insulin-like Growth Factor II mRNA-Binding Protein 3 (IMP3) Expression in Oral Epithelial Dysplasia: a Retrospective Case-Control Study." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1372679866.
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Grenier, Josée. "Effets de différents milieux de culture sur l'expression du gène IGF2, insulin-like growth factor-II, et sur le développement in vitro des embryons bovins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ31731.pdf.
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Palliyaguru, Tishila Sepali. "The expression and regulation of membranetype matrix metalloproteinases (MT-MMPS) in prostate cancer." Thesis, Queensland University of Technology, 2005. https://eprints.qut.edu.au/16009/1/Tishila_Palliyaguru_Thesis.pdf.
Full textAbstract:
Prostate cancer (PCa) represents the most frequently diagnosed cancer and the second leading cause of cancer death in males. Initial development and progression of the disease is mainly regulated by androgens. However, the pathology of the disease may progress to a loss of hormone dependence, resulting in rapid growth and a metastatic phenotype.
Invasion and metastasis of tumour cells results from the degradation of the basement membrane (BM) and extracellular matrix (ECM). The degradation of the BM and ECM is in part mediated by a family of proteinases called the matrix metalloproteinases (MMPs). Currently more than 20 members of the MMP family have been identified and they are further divided in to sub-classes according to their protein structure. Collectively, MMPs are capable of degrading essentially all ECM components. High expression of some MMPs correlates with a malignant phenotype of various tumours.
This study focused on the expression and regulation of a sub-class of MMPs called the membrane-type MMPs (MT-MMPs) in PCa. To date 6 MT-MMPs have been identified and they are characterized by a transmembrane domain, followed by a short cytoplasmic tail (MT1-, MT2-, MT3- and MT5-MMPs) or a glycosylphosphatidylinositol (GPI) moiety (MT4- and MT6-MMPs). MT-MMPs are thought to play a key role in tumour cell invasion by virtue of their ability to activate MMP-2 (a secreted MMP, which is implicated in many metastatic tumours) and their direct degradation activity on ECM components.
Elevated MT-MMP expression has been shown in breast, colon, skin, stomach, lung, pancreas and brain cancers. Until very recently there had been no studies conducted on MT-MMPs in PCa. The few studies preceding or occurring in parallel with this one, have mainly reported the mRNA expression of these enzymes in PCa. Most studies have focused on MT1-MMP. Thus, at the commencement of this project there were many unexplored aspects of the expression and regulation of the broader MT-MMP family in PCa.
The aims of this study were to examine: 1 a) The expression of MT-MMPs in prostate cancer cell lines using RT-PCR and western blot analysis and b) expression of MT1-MMP and MT5-MMP in BPH (benign prostatic hyperplasia) and PCa clinical tissue sections by immunohistochemistry. 2) The regulation of MT1-MMP, MT3-MMP and MT5-MMP in PCa cell lines by Concanavalin A (Con A), phorbol-12-myristate 13-acetate (PMA), dihydrotestosterone (DHT) and insulin-like growth factors I and II (IGF I and IGF II) using western blot analysis.
In this study RWPE1, a transformed but non-tumorigenic prostate cell line was used as a
"normal" prostate cell model, ALVA-41 and LNCaP as androgen-dependent PCa cell models and DU-145 and PC-3 as androgen-independent PCa cell models.
The mRNA expression for the 6 MT-MMPs was determined by RT-PCR. The results indicate that MT1- and MT3-MMP were detected in all cell lines. This is the first study to report MT1-MMP mRNA expression in LNCaP cells and MT3-MMP mRNA in DU-145 cells. MT2-MMP mRNA was detected in only LNCaP and DU-145 cells, whilst MT5-MMP was detected in PC-3, DU-145 and LNCaP cells. nterestingly, MT2-, MT4-, MT5- or MT6-MMP mRNA expression was not detected in the "normal" cell line RWPE1, perhaps indicating an induction in gene transcription in tumour cells. MT4-MMP mRNA was only detected in the androgen-independent cell lines, indicating a potential role in the invasion and metastasis processes of the aggressive androgen-independent PCa. In this study, very low expression of MT6-MMP was detected only in LNCaP and DU-145 cells. Previously there had been no reports on the expression of MT6-MMP in the normal or cancerous prostate.
Due to the mRNA of MT1-, MT3- and MT5-MMPs being the predominant MT-MMPs expressed in the current study, and the availability of suitable antibodies against them, the protein expression of these three MT-MMPs was studied by western blot analysis. MT1-, MT3- and MT5-MMP protein expression was detected in the cell lysates and conditioned medium (CM) of RWPE1, LNCaP and PC-3 cells. For each MT-MMP, various protein species were detected including putative proforms, mature (active) forms, processed or fragmented forms as well as soluble or shed forms. The presence of soluble or shed forms of MT-MMPs in the CM of cultures of "normal" and PCa cells could imply one of the following mechanisms: ectodomain shedding by either extracellular sheddases, the secretion of intracellular processed proteins without the transmembrane domain, the release of membrane vesicles containing membrane-bound enzymes, or the presence of alternatively spliced mRNA, which gives rise to MT-MMPs without a transmembrane
domain. Further characterization of these various forms, including their amino acid
sequence, is required to fully elucidate their structural composition.
Despite the detection of the mRNA, we did not detect the cell-associated proteins of MT1-MMP and MT5-MMP and only very low expression of MT3-MMP in DU-145 cells (CM of DU-145 cells were not screened for soluble forms of the enzymes). This is the first study to report MT5-MMP expression at the protein level in prostate derived cell
lines.
Immunohistochemistry was carried out on benign prostatic hyperplasia (BPH) and PCa clinical tissues using MT1- and MT5-MMP antibodies to determine their cellular localisation in benign and cancer glands. MT1- and MT5-MMPs were expressed in BPH and moderate and high grade PCa. MT1-MMP expression was highest in moderate grade cancer compared to BPH and high grade cancer. MT1-MMP expression was predominantly observed in the cytoplasm of secretory epithelial cells of both benign and cancer glands, although in cancer glands, some nuclear staining was also observed. Stromal expression of MT1-MMP was only observed in high grade cancer.
This study is the first to report the immunolocalization of MT5-MMP outside the brain and in kidneys of diabetic patients. MT5-MMP was predominantly expressed in the cytoplasm of the secretory cells in benign glands. In the cancer glands, staining was heterogeneous with low to intense staining, mainly in the nuclei, plasma membrane and cytoplasm of secretory epithelial cells. Stromal expression of MT5-MMP was only
observed in cancer tissues, particularly in high grade cancer.
To study the regulation of MT-MMPs in PCa, we treated LNCaP and PC-3 cells, with either Con A, PMA, DHT or IGF-I and -II and studied the protein expression of MT1-, MT3- and MT5-MMPs by western blot analysis. Con A and PMA have been shown to stimulate MMP expression in other cell systems.
Con A treatment showed a general increase in the protein expression of MT1-, MT3- and MT5-MMPs. By far the greatest induction by Con A observed was the nearly 4 fold increase in MT5-MMP expression caused by 40μg/mL Con A treatment of PC-3 cells. PMA treatment of LNCaP and PC-3 cells appeared to increase shedding or secretion of all three MT-MMPs in to the CM. This increase in the soluble forms corresponded to a decrease in cell-associated forms in LNCaP cells. Treatment of LNCaP with DHT alone and treatment of LNCaP and PC-3 cells with IGF-I and -II alone failed to detect any change in expression of MT1-MMP.
The information gathered in this study on MT-MMPs with respect to cellular localization,
expression levels and regulation by growth factors or chemicals that mimic their actions,
will aid in our understanding of the role of MT-MMPs in PCa. This study provides strong preliminary data for further research, particularly with respect to functional studies of MT-MMPs in PCa. Understanding the processes which govern the actions of such proteins as these will provide potential insights into development of new management and therapeutic regimens to prevent cancer progression.
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Bekkari, Hicham. "Expression de l'ADNc IGF-II humain (human insulin-like growth factor II) dans des cellules animales recombinantes : purification et caractérisation de la protéine : étude du métabolisme des cellules CHO transfectées." Nancy 1, 1995. http://www.theses.fr/1995NAN10034.
Full textAbstract:
L’objectif de ce travail est l'étude de la production d'un facteur de croissance humain (insulin-like growth factor II) par des cellules animales recombinantes (CHO, chinese hamster ovary). La première partie est consacrée à l'expression de l'ADNc IGF-II dans les cellules CHO-K1. Pour cela, des cellules CHO-K1 ont été cotransfectées par les plasmides pcDNAI-IGF-II et pMAM-néo. Une analyse par dot-blot du milieu conditionné de chaque clone résistant à l'antibiotique G418 a permis de sélectionner les clones les plus producteurs d'IGF-II recombinant. Dans une seconde partie, nous avons mis au point une méthode immunoenzymatique de dosage de l'IGF-2 (ELISA), ce qui nous a permis de sélectionner le clone de cellule CHO le plus producteur d'IGF-II, CHO IGF-II. Cette production est de 400 ng/106cellules/24 h. La stabilité de production de l'IGF-II par les cellules CHO-IGF-II pendant 15 passages, ainsi que la présence de l'ADNc IGF-II dans l'ADN génomique des cellules recombinantes, après analyse par PCR, montrent l'intégration de l’ADNc IGF-II dans les cellules CHO-IGF-II. Dans une troisième partie, les caractéristiques structurales et biologiques de l'IGF-II recombinant ont été étudiées, le facteur de croissance obtenu qui a un poids moléculaire identique à celui de l'IGF-II natif, 7,5 kDa, et une activité biologique sur les cellules MCF-7. La quatrième partie de ce travail est consacrée à l'étude comparée du métabolisme entre les cellules CHO transfectées et non transfectées. Les cellules CHO recombinantes n'ont pas montré de différence dans leurs cinétiques de croissance et du métabolisme par rapport aux cellules sauvages
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Confort, Carole. "Etude d'une forme soluble du récepteur IGFII/M6P dans les cancers du sein." Montpellier 1, 1995. http://www.theses.fr/1995MON1T025.
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Bangcaya, Josette Pesayco. "IGF-I, IGF-II and IGF-IR expression as molecular markers for egg quality in mullet and grouper." Thesis, Queensland University of Technology, 2004. https://eprints.qut.edu.au/16003/1/Josette_Bangcaya_Thesis.pdf.
Full textAbstract:
Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth.
This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success.
Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA.
Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage.
Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.
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Bangcaya, Josette Pesayco. "IGF-I, IGF-II and IGF-IR expression as molecular markers for egg quality in mullet and grouper." Queensland University of Technology, 2004. http://eprints.qut.edu.au/16003/.
Full textAbstract:
Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.
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Neto, João Evangelista Bezerra. "Análise do perfil de expressão de microRNAs em tumores adrenocorticais benignos e malignos humanos." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5166/tde-15082014-153113/.
Full textAbstract:
Introdução: Os mecanismos moleculares que levam ao desenvolvimento de tumores do córtex suprarrenal ainda são pouco compreendidos. Uma alta frequência de carcinomas adrenocorticais na infância tem sido relatada nas regiões sul e sudeste do Brasil, com a presença de uma única mutação germinativa do supressor tumoral p53 (p.R337H) sendo evidenciada em 80- 97% dos casos. Outros fatores implicados na tumorigênese adrenocortical incluem a hiperexpressão das vias IGF2 e Wnt. Os microRNAs, fragmentos de RNA que não codificam proteínas, são capazes de controlar a transcrição gênica exercendo um papel importante no crescimento e proliferação celular. O papel dos microRNA na tumorigênese adrenal ainda não está totalmente elucidado. Objetivos: Avaliar diferenças no perfil de expressão de microRNAs entre tumores benignos e malignos do córtex da suprarrenal da população adulta e pediátrica. Comparar esta expressão entre as amostras caracterizadas pela presença da mutação germinativa p.R337H do supressor tumoral p53, hiperexpressão da via Wnt e da via do IGF2. Métodos: Trinta e seis pacientes não relacionados, adultos e crianças, foram estudados. Os pacientes tiveram avaliação do perfil de produção hormonal e das vias moleculares p53, IGF2 e Wnt. O perfil de expressão de microRNAs foi determinado utilizando-se produto comercial específico TaqMan MicroRNA Human Array (AppliedBiosystems, Forster City, CA, USA). Os dados de expressão foram analisados com o programa Expression Suite (AppliedBiosystems, Forster City, CA, USA) e Realtime Statmainer (Integromics, Granada, Espanha). O estudo de alvos e das redes gênicas afetadas foram estudados com o programa Ingenuity - IPA (Ingenuity, EUA). Resultados: A comparação do perfil de expressão entre adenomas e carcinomas revelou alteração de expressão em 89 e 21 miRNAs em adultos e crianças, respectivamente. Após a correção estatística para múltiplos testes, nove miRNAs mantiveram diferenças significantes em adultos e nenhum em crianças. Dentre os microRNAs com expressão alterada em adultos estavam o miR-483-3p (p=0,011), miR-1290 (p=0,011) e miR-106b (p=0,048). Esses microRNAs foram selecionados para avaliação como biomarcadores por meio de curva ROC. O miR-1290 apresentou o melhor resultado (AUC=1,0; IC 95% 1,0; p=0,003), com valores de expressão de miR-1290 de 10,3 sendo capazes de diferenciar adenomas de carcinomas em adultos com 100% de sensibilidade e especificidade. Na população pediátrica, não foi possível diferenciar adenomas de carcinomas com o uso de microRNAs individuais. A comparação direta entre o perfil de expressão de adenomas da população adulta e pediátrica revelou 38 miRNAs com alteração de expressão. O miR-483-3p e miR-483-5p estavam dentre os mais desregulados e foram os únicos a manter diferença estatística significativa (p=0,009 para ambos), estando hiperexpressos em crianças. A comparação direta do perfil de expressão entre carcinomas da população adulta e pediátrica revelou 26 microRNAs com alteração de expressão, porém sem significância estatística após correção para múltiplos testes. A comparação entre as amostras caracterizadas pela mutação p.R337H do supressor tumoral p53 revelou 53 genes alterados. A comparação entre as amostras caracterizadas por alteração do Wnt revelou 46 genes desregulados. Entretanto, essas alterações não mantiveram significância estatística após correção estatística para múltiplos testes. A comparação entre as amostras caracterizadas por alteração do IGF2 revelou 83 genes alterados, com miR-483-3p (p < 0,001), miR-483-5p (p < 0,001), miR-296-5p (p=0,047) e miR-1290 (p=0,011) mantendo significância estatística após correção para múltiplos testes. O estudo dos potenciais alvos e das redes genicas afetadas pelos miRNAs desregulados observados nesse estudo revelou novas e promissoras vias moleculares que podem ajudar a melhor entender a tumorigenese adrenocortical. Conclusões: Diferenças no perfil de expressão de microRNAs foram observadas entre tumores benignos e malignos do córtex da suprarrenal da população adulta e pediátrica. O ganho de expressão foi o evento mais comum. Os genes miR-483-3p, miR-1290 e miR-106b foram reconhecidos em diversas comparações entre os grupos de interesse e parecem apresentar papel importante na tumorigenese adrenocortical. Além disso, o miR-1290 demonstrou atuar como biomarcador capaz de diferenciar adenomas de carcinomas na população adulta. O estudo de redes gênicas potencialmente afetadas pelos microRNAs que apresentaram alteração de expressão nesse estudo poderá ajudar no melhor entendimento da tumorigênese adrenocorticalIntroduction: The molecular mechanisms that lead to the development of tumors of the adrenal cortex are still poorly understood. A high frequency of pediatric adrenocortical carcinomas has been reported in South and Southeast of Brazil, and a single germline mutation of the tumor suppressor p53 (p.R337H) has been identified in 80-97% of cases. In addition, the overexpression of IGF2 and Wnt pathways are also involved in adrenal tumorigenesis. MicroRNAs, a class of small nonconding RNA, are able to control gene transcription regulating cellular growth and proliferation. However, the role of microRNA has not been fully elucidated in adrenal tumorigenesis. Objectives: To evaluate differences in the expression profile of microRNA between adult and pediatric adrenocortical tumors. To compare microRNA expression profile among samples with and without TP53, Wnt and IGF2 abnormalities. Methods: Thirty-six unrelated patients, adults and children, were studied. Patients had comprehensive hormonal evaluation and tumor samples were studied for TP53, Wnt and IGF2. The expression profile of microRNAs were determined using specific commercial product TaqMan MicroRNA Human Array (AppliedBiosystems, Forster City, CA, USA). The expression data were analyzed with the program Expression Suite (AppliedBiosystems, Forster City, CA, USA) and Realtime Statmainer (Integromics, Granada, Spain). The study of gene networks and affected targets genes have been studied with the Ingenuity program - IPA (Ingenuity, USA). Results: Comparing expression profile between adenomas and carcinomas revealed 89 and 21 deregulated miRNAs in adults and children, respectively. After false discovery rate correction, nine microRNA have maintained significant diferences in miRNAs between adults and none in children. Among microRNAs deregulated in adults were miR-483-3p (p = 0.011), miR-1290 (p = 0.011) and miR-106b (p = 0.048). These microRNAs were selected for evaluation as biomarkers through ROC curve. The miR- 1290 presented the best result (AUC = 1.0; IC 95% 1.0; p = 0.003), with values of expression of miR-1290 of 10.3 being able to differentiate adenomas from carcinomas with 100% sensitivity and specificity. It was not possible to differentiate adenomas from carcinomas by using microRNAs. The direct comparison between the expression profile of adult and pediatric adenomas revealed 38 degulated miRNAs. The miR-483-3p and miR-483-5p were hiperexpressed in children and were the only ones that keept a statistically significant difference (p = 0.009 for both). The direct comparison of the expression profile between adult and pediatric carcinomas revealed 26 deregulated microRNAs, but without statistical significance after correction for multiple testing. The comparison between samples characterized by the p.R337H mutation of tumor suppressor p53 revealed 53 genes deregulated. The comparison between samples characterized by alteration of Wnt reveled 46 microRNAs deregulated. However, after statistical correction for false discovery rate none of them maintained significance. The comparison between samples characterized by change in the IGF2 gene revealed 83 deregulated microRNAs, miR-483-3 p (p < 0.001), miR-483-5 p (p < 0.001), miR-296-5 p (p = 0.047) and miR-1290 (p = 0.011) maintaining statistical significance after correction for false discovery rate. The study of potential targets and molecular networks affected by the deregulatad microRNAs showed promising new molecular pathways that may help better understand the adrenocortical tumorigenese. Conclusions: There were changes in the microRNAs expression profile between malignant and benign tumors of the adrenal cortex of adult and pediatric population. Hyperexpression were the most common presentation. MiR-483-3p, miR-1290 and miR-106b were recognized in various comparisons among groups of interest and appear to have an important role in adrenocortical tumorigenese. In addition, the miR- 1290 can act as a biomarker differentiating adenomas from carcinomas in the adult population. The study of molecular networks potentially affected by the microRNAs deregulated culd contribute to better understanding of adrenocortical tumorigenesis
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Giabicani, Eloïse. "Croissance et système des IGFs (insulin-like growth factors) : l’apport physiopathologique des maladies soumises à empreinte parentale New clinical and molecular insights into Silver–Russell syndrome Roles of Type 1 Insulin-Like Growth Factor (IGF) Receptor and IGF-II in Growth Regulation: Evidence From a Patient Carrying Both an 11p Paternal Duplication and 15q Deletion Diagnosis and Management of Postnatal Fetal Growth Restriction Chromosome 14q32.2 imprinted region disruption as an alternative molecular diagnosis of Silver-Russell Syndrome." Thesis, Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2019SORUS304.pdf.
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La croissance fœtale est sous la dépendance de nombreux facteurs environnementaux, génétiques et hormonaux dont les interactions vont en conditionner le bon déroulement. Le système des insulin-like growth factors (IGFs) joue un rôle prépondérant, à l’interface de ces différents facteurs, pour assurer une bonne croissance fœtale. Dans ce travail, nous nous sommes intéressés aux différents acteurs du système des IGFs dans des pathologies de la croissance fœtale. Dans une approche clinique et expérimentale, nous avons décrit les conséquences fonctionnelles d’anomalies génétiques ou épigénétiques intéressant IGF-I, IGF-II et leur récepteur commun IGF1R. Ainsi, nous avons mis au point un test fonctionnel permettant d’apprécier l’activité in vitro d’IGF1R chez les patients présentant une restriction de croissance fœtale et postnatale. Nous avons également documenté la biodisponibilité d’IGF-I chez des patients présentant un syndrome de Silver-Russell, qui est une pathologie liée à l’empreinte parentale responsable d’une restriction de croissance à début ante-natal. Enfin, nous avons caractérisé le chevauchement clinique et moléculaire entre les patients présentant un SRS ou un syndrome de Temple (autre pathologie liée à l’empreinte parentale), confirmant le rôle prépondérant du défaut d’expression d’IGF2 dans ces deux syndromes. Ces résultats confirment un fonctionnement des gènes soumis à empreinte en réseau et le rôle majeur du système des IGFs dans la croissance fœtale, particulièrement altérée en cas de pathologie intéressant ces gènes soumis à empreinte parentaleFetal growth is dependant of environemental, genetic and hormonal factors which interact to ensure a proper development. Insulin-like growth factors (IGF) system plays a key role in fetal growth by interactions with these differents systems. In this work, we studied the roles of the IGF system in fetal growth restriction diseases. We used both clinical and experimental approaches to enhance knowledge on functional consequences of genetic ou epigenetic defects of IGF system actors. We set-up a functional test to assess IGF1R activity in vitro in patients with restricted fetal and postnatal growth. We also documented the IGF-I bioavailability in patients with Silver-Russell syndrome, which is an imprinting disorder responsible for fetal and postnatal growth restriction. We characterized the clinical and molecular overlap of Silver-Russell and Temple syndrome (another imprinting disease affecting growth and metabolism) and confirmed the central role of IGF2 in the physiopathology of these disorders. These results confirmed the integration of imprinted genes in a large co-regulation network and the major role of IGF system actors in fetal growth which is usually impaired when these imprinted genes are affected
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Palliyaguru, Tishila Sepali. "The expression and regulation of membranetype matrix metalloproteinases (MT-MMPS) in prostate cancer." Queensland University of Technology, 2005. http://eprints.qut.edu.au/16009/.
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Prostate cancer (PCa) represents the most frequently diagnosed cancer and the second leading cause of cancer death in males. Initial development and progression of the disease is mainly regulated by androgens. However, the pathology of the disease may progress to a loss of hormone dependence, resulting in rapid growth and a metastatic phenotype.
Invasion and metastasis of tumour cells results from the degradation of the basement membrane (BM) and extracellular matrix (ECM). The degradation of the BM and ECM is in part mediated by a family of proteinases called the matrix metalloproteinases (MMPs). Currently more than 20 members of the MMP family have been identified and they are further divided in to sub-classes according to their protein structure. Collectively, MMPs are capable of degrading essentially all ECM components. High expression of some MMPs correlates with a malignant phenotype of various tumours.
This study focused on the expression and regulation of a sub-class of MMPs called the membrane-type MMPs (MT-MMPs) in PCa. To date 6 MT-MMPs have been identified and they are characterized by a transmembrane domain, followed by a short cytoplasmic tail (MT1-, MT2-, MT3- and MT5-MMPs) or a glycosylphosphatidylinositol (GPI) moiety (MT4- and MT6-MMPs). MT-MMPs are thought to play a key role in tumour cell invasion by virtue of their ability to activate MMP-2 (a secreted MMP, which is implicated in many metastatic tumours) and their direct degradation activity on ECM components.
Elevated MT-MMP expression has been shown in breast, colon, skin, stomach, lung, pancreas and brain cancers. Until very recently there had been no studies conducted on MT-MMPs in PCa. The few studies preceding or occurring in parallel with this one, have mainly reported the mRNA expression of these enzymes in PCa. Most studies have focused on MT1-MMP. Thus, at the commencement of this project there were many unexplored aspects of the expression and regulation of the broader MT-MMP family in PCa.
The aims of this study were to examine: 1 a) The expression of MT-MMPs in prostate cancer cell lines using RT-PCR and western blot analysis and b) expression of MT1-MMP and MT5-MMP in BPH (benign prostatic hyperplasia) and PCa clinical tissue sections by immunohistochemistry. 2) The regulation of MT1-MMP, MT3-MMP and MT5-MMP in PCa cell lines by Concanavalin A (Con A), phorbol-12-myristate 13-acetate (PMA), dihydrotestosterone (DHT) and insulin-like growth factors I and II (IGF I and IGF II) using western blot analysis.
In this study RWPE1, a transformed but non-tumorigenic prostate cell line was used as a
"normal" prostate cell model, ALVA-41 and LNCaP as androgen-dependent PCa cell models and DU-145 and PC-3 as androgen-independent PCa cell models.
The mRNA expression for the 6 MT-MMPs was determined by RT-PCR. The results indicate that MT1- and MT3-MMP were detected in all cell lines. This is the first study to report MT1-MMP mRNA expression in LNCaP cells and MT3-MMP mRNA in DU-145 cells. MT2-MMP mRNA was detected in only LNCaP and DU-145 cells, whilst MT5-MMP was detected in PC-3, DU-145 and LNCaP cells. nterestingly, MT2-, MT4-, MT5- or MT6-MMP mRNA expression was not detected in the "normal" cell line RWPE1, perhaps indicating an induction in gene transcription in tumour cells. MT4-MMP mRNA was only detected in the androgen-independent cell lines, indicating a potential role in the invasion and metastasis processes of the aggressive androgen-independent PCa. In this study, very low expression of MT6-MMP was detected only in LNCaP and DU-145 cells. Previously there had been no reports on the expression of MT6-MMP in the normal or cancerous prostate.
Due to the mRNA of MT1-, MT3- and MT5-MMPs being the predominant MT-MMPs expressed in the current study, and the availability of suitable antibodies against them, the protein expression of these three MT-MMPs was studied by western blot analysis. MT1-, MT3- and MT5-MMP protein expression was detected in the cell lysates and conditioned medium (CM) of RWPE1, LNCaP and PC-3 cells. For each MT-MMP, various protein species were detected including putative proforms, mature (active) forms, processed or fragmented forms as well as soluble or shed forms. The presence of soluble or shed forms of MT-MMPs in the CM of cultures of "normal" and PCa cells could imply one of the following mechanisms: ectodomain shedding by either extracellular sheddases, the secretion of intracellular processed proteins without the transmembrane domain, the release of membrane vesicles containing membrane-bound enzymes, or the presence of alternatively spliced mRNA, which gives rise to MT-MMPs without a transmembrane
domain. Further characterization of these various forms, including their amino acid
sequence, is required to fully elucidate their structural composition.
Despite the detection of the mRNA, we did not detect the cell-associated proteins of MT1-MMP and MT5-MMP and only very low expression of MT3-MMP in DU-145 cells (CM of DU-145 cells were not screened for soluble forms of the enzymes). This is the first study to report MT5-MMP expression at the protein level in prostate derived cell
lines.
Immunohistochemistry was carried out on benign prostatic hyperplasia (BPH) and PCa clinical tissues using MT1- and MT5-MMP antibodies to determine their cellular localisation in benign and cancer glands. MT1- and MT5-MMPs were expressed in BPH and moderate and high grade PCa. MT1-MMP expression was highest in moderate grade cancer compared to BPH and high grade cancer. MT1-MMP expression was predominantly observed in the cytoplasm of secretory epithelial cells of both benign and cancer glands, although in cancer glands, some nuclear staining was also observed. Stromal expression of MT1-MMP was only observed in high grade cancer.
This study is the first to report the immunolocalization of MT5-MMP outside the brain and in kidneys of diabetic patients. MT5-MMP was predominantly expressed in the cytoplasm of the secretory cells in benign glands. In the cancer glands, staining was heterogeneous with low to intense staining, mainly in the nuclei, plasma membrane and cytoplasm of secretory epithelial cells. Stromal expression of MT5-MMP was only
observed in cancer tissues, particularly in high grade cancer.
To study the regulation of MT-MMPs in PCa, we treated LNCaP and PC-3 cells, with either Con A, PMA, DHT or IGF-I and -II and studied the protein expression of MT1-, MT3- and MT5-MMPs by western blot analysis. Con A and PMA have been shown to stimulate MMP expression in other cell systems.
Con A treatment showed a general increase in the protein expression of MT1-, MT3- and MT5-MMPs. By far the greatest induction by Con A observed was the nearly 4 fold increase in MT5-MMP expression caused by 40μg/mL Con A treatment of PC-3 cells. PMA treatment of LNCaP and PC-3 cells appeared to increase shedding or secretion of all three MT-MMPs in to the CM. This increase in the soluble forms corresponded to a decrease in cell-associated forms in LNCaP cells. Treatment of LNCaP with DHT alone and treatment of LNCaP and PC-3 cells with IGF-I and -II alone failed to detect any change in expression of MT1-MMP.
The information gathered in this study on MT-MMPs with respect to cellular localization,
expression levels and regulation by growth factors or chemicals that mimic their actions,
will aid in our understanding of the role of MT-MMPs in PCa. This study provides strong preliminary data for further research, particularly with respect to functional studies of MT-MMPs in PCa. Understanding the processes which govern the actions of such proteins as these will provide potential insights into development of new management and therapeutic regimens to prevent cancer progression.
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Cripps, Joanna Elizabeth. "The expression of ovine IGF II mRNA during embryonic development." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321106.
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Almeida, Madson Queiroz de. "Expressão dos genes IGF-II, IGF-IR, SF-1 e DAX-1 em tumores adrenocorticais de crianças e adultos." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-31102008-153148/.
Full textAbstract:
Introdução: A patogênese molecular dos tumores adrenocorticais é heterogênea e ainda pouco compreendida. A hiperexpressão do gene do fator de crescimento semelhante à insulina II (IGF-II) tem sido demonstrada na maioria dos carcinomas adrenocorticais em adultos. Os efeitos mitogênicos do IGF-II são mediados pela interação com o receptor de IGF-I (IGF-IR). Adicionalmente, o fator esteroidogênico 1 (SF-1) e o fator codificado por uma região crítica do cromossomo X associada ao sexo reverso e à hipoplasia adrenal congênita (DAX-1), ambos envolvidos no desenvolvimento e na esteroidogênese adrenal, também têm sido implicados na tumorigênese adrenocortical. Objetivos: Analisar a expressão gênica e a imunorreatividade dos fatores IGF-II, IGF-IR, SF-1 e DAX-1 em tumores adrenocorticais de crianças e adultos. Avaliamos ainda os efeitos de um inibidor seletivo do IGF-IR (NVP-AEW541) na proliferação celular e apoptose de linhagens celulares de tumores adrenocorticais. Métodos: Neste estudo, a expressão gênica foi determinada por PCR quantitativa em tempo real em 57 tumores adrenocorticais (37 adenomas e 20 carcinomas). Vinte e três pacientes tinham idade inferior ou igual a 15 anos. A análise de imunohistoquímica foi realizada em 109 tumores adrenocorticais (71 adenomas e 38 carcinomas). Os efeitos do tratamento com NVP-AEW541 (0,3 a 30 M) na proliferação celular e apoptose foram avaliados nas células NCI H295 de carcinoma adrenocortical humano e em uma nova linhagem celular estabelecida a partir de um adenoma adrenocortical pediátrico da nossa casuística. Resultados: A hiperexpressão do gene IGF-II foi evidenciada nos tumores adrenocorticais benignos e malignos de crianças (média ± EPM, 50,8 ± 18,5 vs. 31,2 ± 3,7, respectivamente; p= 0,23). Em adultos, a expressão do gene IGF-II foi significativamente mais elevada nos carcinomas adrenocorticais quando comparada com os adenomas (270,5 ± 130,2 vs. 16,1 ± 13,3; p= 0,0001). O percentual das células neoplásicas imunorreativas para o IGF-II não foi significativamente diferente entre os adenomas e carcinomas adrenocorticais pediátricos (14,1 ± 2,8% vs. 31,1 ± 13,1%, respectivamente; p= 0,32). Em adultos, o percentual das células neoplásicas positivas para o IGF-II foi significativamente maior nos carcinomas adrenocorticais em relação aos adenomas (34,4 ± 5,8% vs. 14,2 ± 3,2, respectivamente; p= 0,03). Os valores de RNAm do IGF-IR foram significativamente mais elevados nos carcinomas adrenocorticais pediátricos em relação aos adenomas (9,1 ± 1,2 vs. 2,6 ± 0,3; p= 0,0001), enquanto a expressão deste receptor foi similar nos tumores adrenocorticais benignos e malignos de adultos (1,6 ± 0,3 vs. 1,8 ± 0,5, respectivamente; p= 0,75). Os valores de RNAm do IGF-IR [risco relativo (RR) 2,0, intervalo de confiança (IC) de 95% 1,2 a 3,1; p= 0,004] e os critérios histopatológicos de Weiss (RR 1,8, IC de 95% 1,2 a 2,7; p= 0,003) foram marcadores independentes de metástases em crianças e adultos, respectivamente. O NVP-AEW541 inibiu a proliferação celular estimulada por IGF-II de forma dose e tempo dependentes nas 2 linhagens celulares de tumores adrenocorticais através de uma significativa indução da apoptose. Adicionalmente, a hiperexpressão do gene SF-1 foi identificada em 13% e 15% dos tumores adrenocorticais diagnosticados em crianças e adultos, respectivamente. O percentual das células neoplásicas com imunorreatividade nuclear para SF-1 foi significativamente maior nos tumores adrenocorticais pediátricos em relação aos tumores diagnosticados em adultos (29,1 ± 5,4% vs. 8,3 ± 2,3%, respectivamente; p= 0,0001). Estes dados indicam que o aumento da expressão do SF-1 nos tumores adrenocorticais pediátricos ocorre em nível pós-traducional. A hiperexpressão do gene DAX-1 foi identificada em 39% dos tumores adrenocorticais, com uma prevalência semelhante em crianças e adultos. De forma similar, o aumento da expressão da proteína DAX-1 foi identificado em 36% e 27% dos tumores adrenocorticais diagnosticados em crianças e adultos, respectivamente. A imunorreatividade nuclear para DAX- 1 foi semelhante nos adenomas e carcinomas adrenocorticais (29,2 ± 3,8% vs. 21,4 ± 5,8% das células neoplásicas, respectivamente; p= 0,12). Conclusões: A hiperexpressão do IGF-II tem um papel relevante na tumorigênese adrenocortical. A hiperexpressão do gene IGF-IR foi um marcador biológico independente do carcinoma adrenocortical metastático em crianças. Os efeitos anti-tumorais in vitro do NVP-AEW541 sugerem que o IGF-IR constitui um potencial alvo terapêutico para o carcinoma adrenocortical humano. Adicionalmente, o aumento da expressão do SF-1 foi evidenciado predominantemente nos tumores adrenocorticais pediátricos. A hiperexpressão do DAX-1 constitui um evento importante na patogênese molecular dos tumores adrenocorticais benignos e malignosIntroduction: The molecular pathogenesis of adrenocortical tumors is heterogeneous and incompletely understood. Insulin-like growth factor II (IGF-II) overexpression has been demonstrated in adult adrenocortical carcinomas. IGF-II exerts its mitogenic effects through interaction with IGF-I receptor (IGF-IR). In addition, steroidogenic factor 1 gene (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene (DAX-1), which regulate adrenal development and steroidogenesis, have been also involved in adrenocortical tumorigenesis. Objectives: To analyze gene and protein expression of IGF-II, IGF-IR, SF-1 and DAX-1 in pediatric and adult adrenocortical tumors. We also evaluated the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cell lines. Methods: Gene expression was determined by quantitative real-time PCR in 57 adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults. Twenty and three patients were younger than 15 years. A tissue microarray analysis was performed on a large cohort of 109 ACT (71 adenomas and 38 carcinomas; 39 children and 70 adults) In addition, the effects of NVP-AEW541 treatment (0.3 to 30M) on proliferation and apoptosis were investigated in the NCI H295 cell line and in a new cell line established from a pediatric adrenocortical adenoma of our cohort. Results: IGF-II transcripts were overexpressed in pediatric adrenocortical carcinomas and adenomas (mean ± SE, 50.8 ± 18.5 vs. 31.2 ± 3.7, respectively; p= 0.23). IGF-II gene expression was significantly higher in adult adrenocortical carcinomas than in adenomas (270.5 ± 130.2 vs. 16.1 ± 13.3; p= 0.0001). The percentual of neoplastic cells immunostaining for IGF-II was not statistically different between pediatric adrenocortical adenomas and carcinomas (14.1 ± 2.8% vs. 31.1 ± 13.1%, respectively; p= 0.32). Otherwise, the percentual of positive neoplastic cells for IGF-II was significantly higher in adult adrenocortical carcinomas than in adenomas (34.4 ± 5.8% vs. 14.2 ± 3.2, respectively; p= 0.03). IGF-IR mRNA levels were significantly higher in pediatric adrenocortical carcinomas than in adenomas (9.1 ± 1.2 vs. 2.6 ± 0.3; p= 0.0001), whereas similar IGF-IR expression levels were identified in adult adrenocortical carcinomas and adenomas (1.6 ± 0.3 vs. 1.8 ± 0.5, respectively; p= 0.75). In a Cox multivariate analysis, IGF-IR gene expression [hazard ratio (HR) 2.0, 95% confidence interval (CI) 1.2 to 3.1; p= 0.004] and Weiss score (HR 1.7, 95% CI 1.2 to 2.7; p= 0.003) were independent biomarkers of metastasis in pediatric and adult adrenocortical tumors, respectively. Furthermore, NVP-AEW541 blocked cell proliferation in a dose- and time-dependent manner in both NCI H295 and pediatric adrenocortical cell lines through a significant increase of apoptosis. Additionally, SF-1 gene overexpression was identified in 13% and 15% of pediatric and adult adrenocortical tumors, respectively. The percentual of neoplastic cells with nuclear immunoreactivity for SF-1 was significantly higher in pediatric than in adult adrenocortical tumors (29.1 ± 5.4% vs. 8.3 ± 2.3%, respectively; p= 0.0001). These findings suggest that SF-1 overexpression occurs at the translational level in pediatric adrenocortical tumors. DAX-1 gene overexpression was identified in 39% of adrenocortical tumors with a similar frequency in children and adults. Similarly, DAX-1 protein overexpression was identified in 36% and 27% of pediatric and adult adrenocortical tumors, respectively. DAX-1 immunostaining on nuclei was not statistically different in benign and malignant adrenocortical tumors (29.2 ± 3.8% vs. 21.4 ± 5.8% of neoplastic cells, respectively; p= 0.12). Conclusion: IGF-II overexpression has a pivotal role to adrenocortical tumorigenesis. IGFIR overexpression was a potential biomarker of metastases in children with adrenocortical carcinoma. We demonstrated that a selective IGF-IR kinase inhibitor had anti-tumor effects in adult and pediatric ACT cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma. In addition, SF-1 overexpression might be mainly involved in pediatric adrenocortical tumorigenesis. DAX-1 overexpression has an important role to molecular pathogenesis of benign and malignant adrenocortical tumors
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Martin, Katrin. "Untersuchungen der Insulinähnlichen Wachstumsfaktoren IGF-I und IGF-II, deren Bindeproteine IGFBP-2 und IGFBP-3 und der Säurelabilen Untereinheit ALS bei Kindern mit soliden Tumoren." [S.l. : s.n.], 2007.
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Narayanan, Ram. "Genotype and phenotype interactions of the insulin-like growth factor system in type 2 diabetes." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/genotype-and-phenotype-interactions-of-the-insulinlike-growth-factor-system-in-type-2-diabetes(5e6925fb-195d-47d8-a06d-8957a8f3b86f).html.
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Background: Multiple lines of evidence implicate the insulin-like growth factor(IGF) group of proteins in human type 2 diabetes. The actions of IGF-I and IGF-IIare modulated through their interaction with IGF binding proteins. A holisticapproach to study the IGF system is preferable to analyses of individual proteininteractions as the inter-relationships between these proteins are complex. Inparticular, the associations of IGF-II and its associated binding proteins withcardiovascular risk have been inadequately studied. This study aimed to study indetail the genotype and phenotype interactions of the IGF system with longitudinalcardiovascular risk factor trends and phenotypic outcomes in type 2 diabetes.Methods: 1000 subjects of predominantly Caucasian origin from the SalfordDiabetes Cohort were studied. Measurements of IGF proteins (IGF-I, IGF-II,IGFBP-1, IGFBP-2 and IGFBP-3) were performed in 554 of these patients. 991Caucasian subjects were successfully genotyped for 76 single nucleotidepolymorphisms (SNPs) related to ten genes in the IGF system. In this project weanalysed associations of the studied SNPs with the measured IGF proteins as well aslongitudinal risk factor trends. In addition, the baseline concentrations of themeasured proteins were studied for associations with cardiovascular risk factortrends and vascular outcomes.Results: This project demonstrates for the first time that high serum IGF-IIconcentration at baseline predicts longitudinal increases in high-density lipoproteincholesterol. High baseline IGF-II was also observed to predict longitudinal weightloss. High baseline concentration of IGFBP-2 (which has a preferential associationof IGF-II over IGF-I) was associated with a number of favourable longitudinalcardiovascular risk trends like increased HDL cholesterol and decreased diastolicblood pressure. However high IGFBP-2 was also associated with deterioration inrenal function and increased all-cause and cardiovascular mortality. The IGF2 geneand the genes encoding IGFBP-2 and IGFBP-5 (proteins with IGF-II bindingaffinity) were also associated with longitudinal trends in renal function, bloodpressure and cholesterol concentration.Discussion: This study is the most detailed exploration to date of the genotype andphenotype interactions of the IGF system in a Caucasian population with type 2diabetes. Results from this study strongly hint that changes in IGF-II bioavailabilitymay influence inter-individual variations in cardiovascular risk. The precisebiological role of IGF-II merits clarification in future expression studies in renal,adipose and vascular tissues. Replication of significant results in an independentdiabetes cohort and measurement of other IGF binding proteins will be performed inthe next stage of this study.
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Soares, Fernando Peixoto. "Avaliação de componentes da matriz extracelular na reparação de defeitos de furca classe II após o enxerto de tecido reparativo de alvéolos dentários tratados com fatores de crescimento." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/23/23146/tde-28092009-153134/.
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O tecido reparativo de alvéolos de extração foi proposto como material de enxerto no tratamento de defeitos periodontais e a adição de fatores de crescimento aos alvéolos de extração melhoraria o potencial regenerativo deste tecido quando utilizado como enxerto. O objetivo deste trabalho foi avaliar qualitativamente, por meio de análise imuno-histoquímica, a reparação de defeitos agudos de furca classe II após o enxerto deste tecido. Foram extraídos os segundos e terceiros pré-molares superiores de 4 cães. Nos alvéolos resultantes foram aplicados fator de crescimento derivado de plaquetas-BB (PDGF-BB) e fator de crescimento semelhante à insulina-I (IGF-I), na concentração de 6 !g/ml cada. Após cinco dias, 24 defeitos agudos (12 controles e 12 testes) foram criados nos segundos, terceiros e quartos pré-molares inferiores. Apenas os sítios teste receberam o enxerto. Os retalhos foram posicionados coronariamente em ambos os lados e suturados. Após 45 dias, os espécimes foram analisados, em um plano vestíbulo-lingual, por meio de técnica imuno-histoquímica para osteopontina (OPN), sialoproteína óssea (BSP) e osteonectina (ONC). Nos tecidos periodontais originais, a marcação para os anticorpos testados foi fracamente positiva para a matriz extracelular (MEC), caracterizando a presença de tecidos maduros. Já no interior dos defeitos, houve diferença na marcação entre grupos, com marcação mais pronunciada para BSP e OPN no grupo controle, evidenciando uma maior atividade metabólica neste grupo, sugerindo que os tecidos reparativos do grupo teste já se encontravam em uma fase mais avançada do processo de reparação.The tissue from healing extraction sockets is considered an excellent bone grafting material in the therapy of periodontal defects and the association of growth factors to the extraction sockets would increase the regenerative potential of this tissue when utilized as a bone graft. The aim of this investigation was to evaluate qualitatively the healing of acute class II furcation defects when grafted with granulation tissue from healing extraction sockets previously treated with an association of platelet derived growth factor-BB (PDGF-BB) and insulin-like growth factor-I (IGF-I), both in a concentration of 6 !g/ml. The second an third upper premolars of four dogs were extracted and the growth factors were applied into the sockets. After a healing period of five days, 24 class II furcation defects (12 tests and 12 controls) were surgically created at the buccal surface of the second, third and fourth mandibular premolars. The 12 experimental sites were grafted with the tissue from the healing sockets, while the 12 control sites healed without any graft. The flaps were coronally positioned and sutured. After 45 days, tissues were analyzed by immunohistochemistry for osteopontina (OPN), bone sialoprotein (BSP) and osteonectina (ONC), in a buccal-lingual plane. Histologically, healing occurred similarly in both groups. Pristine periodontal tissues extracellular matrix stained faintly for the tested antibodies, which characterized the presence of mature tissues. The staining of test and control healing tissues showed a more intense metabolic activity of the control group. This fact suggests that the tissues in the treated defect of test group were already in a more advanced phase of the repair process.
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Jiang, Cheng-Shian, and 江承先. "Production of insulin-like growth factor II feed yeast." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/72693283631352820170.
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碩士國立臺灣大學
微生物與生化學研究所
93
Feed yeast Candida utilis BCRC 21645 was used as a host for tilapia insulin-like growth factor 2 expression. An expression plasmid pGAPZB-IGF2 was constructed by inserting igf2 gene into a commercial vector pGAPZB belonging to Pichia pastoris expression system, because a commercial vector for C. utilis was not available and P. pastoris is more relative to C. utilis than Saccharomyces cerevisiae. The expression plasmid pGAPZB-IGF2 was transferred into C. utilis for the expression of tilapia igf2 gene. The recombinant IGF2 was produced as soluble protein, and its content was as high as 6.7% of the soluble protein (Hu, 2004). After six months of storage, IGF2 was not produced in C. utilis/pGAPZB-IGF2 . After PCR analysis, we found that a fragmaent of GAP promoter was lost in chromosome DNA of transformants. The expression plasmid pGAPZB-IGF2 was prepared from Escherichia coli BL21(DE3) and linearized by Bgl II. Linearized pGAPZB-IGF2 was used to transform C. utilis by electroporation . The maximum transformation efficiency obtained was 16 transformants per μg of linearized pGAPZB-IGF2 at a field strength of 7.5 kv cm-1, an internal resistance of 800 Ω and 10 μg plasmid DNA. Although the DNA sequencing of PCR analysis demonstrated that linearized pGAPZB-IGF2 was successfully insert into chromosome DNA of C. utilis, igf2 gene was not expressed in transformants. There is no homologous sequence in pGAPZB-IGF2 result in the low transformation efficiency. We didn’t comfirm if the GAP promoter of P. pastoris work in C. utilis. The difference of GAP promoter between C. utilis and P. pastoris or the codon usage of structure gene in host may be concerned to explain why no igf2 gene expression in transformants. The transformants constructed before may be an exception of unstable insertion of igf2 gene, the expression of igf2 gene was not initialized by GAP promoter in expression plasmid pGAPZB-IGF2. We suggested to construct an expression plasmid with the homologous DNA sequence of C. utilis such as GAP promoter, rDNA gene or URA3 gene for the improvement of transformation efficiency and the expression of heterologous gene.
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Mörth, Corinna [Verfasser]. "Consequences of postnatal insulin-like growth factor II overexpression in insulin-like growth factor I deficient mice / by Corinna Mörth." 2005. http://d-nb.info/978816617/34.
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You, Hui-Ling, and 尤慧玲. "Overexpression of serum insulin-like growth factor (IGF-II) in." Thesis, 1998. http://ndltd.ncl.edu.tw/handle/33300417610019712769.
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碩士高雄醫學院
生物化學研究所
86
Hepatocellular carcinoma (HCC) is one of the most common and devastating human malignant tumors. Although hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the major risk factors for HCC, the mechanisms of hepatocarcinogenesis have not yet clearly understood. Recently, a growing body of evidence demonstrate the importance of peptide growth factors, their receptors and related proteins in the process of malignant transformation. The unregulated expression of growth factors or components of thProliferation of neoplastic and non-neoplastic cells is determined by a complex network of growth factors cooperating as stimulatory or inhibitory modulators of growth in an autocrine or paracrine manner. The characterization of the complete network of growth factors active in a particular tumor type, therefore, might provide important insights into the mechanisms determining its biological behavior and eventually facilitate the design of novel concepts in tumor therapy. Growth factors supposedly also affecOver-expression of oncogenes or growth factor genes and inactivation of tumor suppressor gene are characteristic finding in human HCCs. In addition, several reports indicate the regulation of oncogene and/or growth factor expression by HBV and/or HCV in human HCC. However, the serum expression of insulin-like growth factor-II (IGF-II) in patients with HCC remains controversy. Furthermore, the interaction between IGF-II and transforming growth factor-β 1 (TGF-β 1 ) through IGF receptor type II ( IGF2R, a tTo investigate the serum insulin-like growth factor-II (IGF-II) level in relation to HCC related to HBV and HCV infection, serum IGF-II (by immunoradiometric assay), HBV-DNA and HCV-RNA (by polymerase chain reaction), and prealbumin (to adjust nutritional status) were measured in 138 patients with HCC, 53 patients with cirrhosis, 56 patients with chronic hepatitis (CH), and 87 healthy controls. With nutritional adjustment, serum IGF-II level in controls was significantly lower than those in patients with CH To investigate the modulation of IGF axis and TGF-β1 in expeimental hepatocarcinogenesis. Cirrhosis, preneoplastic nodules and/or HCC were induced in rats by carbon tetrachloride and diethylnitrosamine.We performed immunohistochemical staining of IGFBP3, Ras, cPKC a, IGF-II and TGF-β1, RT-PCR for IGF-II and TGF-β1 mRNA determination, cross-link affinity labeling for IGF2R measure, and western blot for IGF1R, IGFBP3 and TGF -β receptors . The results are as follows: IGF-II and TGF-β1 were absent in noOur results indicate that modulation between IGF axis and TGF-β 1 plays an important role during hepatocarcinogenesis and growth control of HCC cells. There is overexpression of IGF-II in the serum of patients with HCC. The conclusion is as follows: IGF-II plays an important role in the proliferation and differentiation of both benign and malignant hepatocytes.1 Over-expression of IGF-II and IGFBP3 (to prolong half life of IGF-II) during hepatic carcinogenesis indicates a role of this mitogen in neoplastic transformation and proliferation of cancer cells during hepatocarcinogenesis.2 Expression of tyrosine kinase-containing IGF1R activates IGF-II during tumorigenesis.3 Inactivation of IGF2R gene, as evidenced by lower expression of IGF2R in HCC, causes both diminished growth suppression (via decreased activation of TGF-β1) and augmented growth stimulation (via decreased degradation of the IGF-II).4 Over-expression of ras and cPKC in preneoplastic nodules and HCC promote tumorigenesis.
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Jung, Liu Chung, and 劉忠榮. "The Role of Insulin-Like Growth Factor-II in the Synergistic Effect of Cardiomyocytes Apoptosis Induced by Angiotensin-II and Insulin-Like Growth Factor-I Resistance." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/32841015125859009597.
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碩士中山醫學大學
生物化學研究所
90
Apoptosis of cardiac myocytes is one of the cause of heart failure. Hypertension and diabetes being able to lead to cardiomyocytes apoptosis, might eventually develop into heart failure. The main purpose of this study is to identify under the condition of hypertension induced by Angiotensin-II (Ang-II) whether the reduction of insulin-like growth factor-I (IGF-I) through aging and / or insulin-like growth factor-I receptor (IGF-IR) resistant of diabetic mellitus might further enhance heart failure.Applying primary cardiomyocytes from adult rat hearts and transforming embryonic heart cells of rat, H9C2 with serum-free medium, we added Ang-II with or without antisense IGF-I or IGF-IR antibodies to identify the heart failure-enhancing effects. Results show that with AngII treatment and IGF-I deficiencies induced both cardiomyocytes hypertrophy and apoptosis.An enlarged size、DNA fragmentation、nuclear condensation and elevating pro-apoptotic proteins, such as Bad and cytochrome c, appeared in both primary cardiomyocytes or H9C2. Particularly, the pro-apoptotic effects of Ang-II were augmented by IGF-I deficiency and anti-IGF-IR treatment. Insterestingly, the apoptosis and hypertrophy of cardiomyocytes, the increase of Bad protein and the release of cytochrome c were all reversed by antisense IGF-II or anti-IGF-II treatment. At the mean time, the upregulation of IGF-II and IGF-II receptor gene expression and protein production were both observed under IGF-I deficient and IGF-I receptor resistant. Taken together, our results suggested that cardiomyocyte hypertrophy and apoptosis caused by Ang-II were augmented by IGF-I deficiency and/or IGF-IR resistant, which might result comes from IGF-II gene up-regulation.
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Huang, Shi Xin, and 黃世心. "Influence of insulin-like growth factor II antisense oligonucleotides on growth of hepatoma cell lines." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/88399982986812334284.
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碩士國立臺灣大學
醫事技術研究所
83
Insulin-like growth factor II (IGF-II) is a single chain poly- peptide growth factor which is structurally related to insulin and insulin-like growth factor type I (IGF-I).IGF-II is thought to be involved in fetal tissues development and growth. Many papers have showed that the expression of IGF-II mRNA is markedly elevated in several human fetal and tumor tissues. It is suggested that the growth factor might be involved in cancer progression by an autocrine/ paracrine mechanism. The main purpose of the present study was to characterize the role of IGF-II in hepatoma cell lines growth. We set up an enzyme immunoassay system to determinate the IGF-II protein levels of several hepatoma cell lines and added IGF-II to cell culture system to see the mitogenic effect of IGF-II. We also constructed a vector which contained IGF-II cDNA and SP6 / T7 promoters, then synthesized antisense oligonucleotides which are complementary to IGF-II mRNA. We applied antisense oligo- nucleotides in this in vitro transcription/ translation system. After screening useful antisense oligonucleotides in vitro, we utilized cell culture system to inhibit IGF-II production and evaluated cell growth rate to find the relationship between IGF-II and cell growth. Finally, we subcutaneously injected HepG2 cells and antisense oligonucleotides to SCID mice and observed tumorigenesis progression whether it could be delayed by inject-ing antisense oligonucleotides. We found that antisense oligonucleotides can inhibit IGF-II production. Cell growth rate would slow down when IGF-II pro- duction was inhibited by antisense oligonucleotides. This finding suggested that IGF-II could be an autocrine/ paracrine growth factor of hepatoma HepG2 cells.