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研究生: 賴怡君
Yi-Chun Lai
論文名稱: KLK1、TIEG1/EGR alpha、TNF-alpha、IL-1 alpha及IL-1 beta基因多型性與原發性膀胱輸尿管逆流疾病發生及腎病進行的相關性研究
Association of KLK1, TIEG1/EGR alpha, TNF-alpha, IL-1 alpha and IL-1 beta Gene Polymorphisms with Primary VUR Development and Progression
指導教授: 李桂楨
Lee, Guey-Jen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2004
畢業學年度: 92
語文別: 中文
論文頁數: 65
中文關鍵詞: 基因多型性原發性膀胱輸尿管逆流疾病發生腎病進行腫瘤壞死因子介白素1
英文關鍵詞: Gene Polymorphism, Primary VUR, disease development and progression, KLK1, TIEG1, EGR alpha, TNF-alpha, IL-1
論文種類: 學術論文
相關次數: 點閱:214下載:2
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  • 膀胱輸尿管逆流(VUR)為常見的小兒先天性泌尿道疾病,部份與家族性遺傳相關,但目前致病之基因仍不明確,且VUR孩童的疾病進展或預後情況不一,嚴重者會進入末期腎病(ESRD)。本研究即在探討人類腎臟KLK1、TIEG1/EGR、TNF-、IL-1、IL-1基因的多型性與台灣孩童VUR發生及腎病進行的相關性。KLK1的啟動子中-130 GN多型性係藉genotyping、SSCP及allele-specific PCR來分析基因型。TIEG1/EGR基因經由直接定序及比對,找出兩個尚未報導過的多型點:EGR啟動子上的C-1445G,及兩基因共用的第三外顯子上的T216 (A>C),此二多型性點與TNF-的C-863A、C-857T及IL-1的C-889T、IL-1的C-511T皆藉由限制酶切割PCR放大片段來分析基因型,另IL-1的3'UTR多型性則利用SSCP的方式區分基因型。結果顯示EGR的-1445C與VUR疾病的發生有關,而與腎病進行有相關的多型性則有兩個,分別為KLK1的啟動子的-130 GN多型性的K對偶基因及TNF-的-863A多型性,其他多型性則未發現與VUR疾病的發生或腎病進行有關。本實驗尚有做KLK1及EGR啟動子多型性轉錄活性分析,以報導基因luciferase於HEK-293細胞株中進行基因表現,結果顯示KLK1 -130 GN多型性的K對偶基因及EGR多型性的G對偶基因有較低的轉錄活性。

    Vesicoureteral reflux (VUR) is a common pediatric disease. The development of VUR is highly familially inherited and the disease progression is variable. Some patients may lead to severe end-stage renal disease (ESRD). In this study, polymorphisms of human renal kallikrein gene (KLK1), TGFβ-inducible early gene 1/early growth response  gene (TIEG1/EGR), tumor necrosis factor- gene (TNF-), interleukin-1 gene (IL-1), interleukin-1 gene (IL-1) were evaluated for the association with VUR development and progression in Taiwanese children. The KLK1 promoter -130 GN polymorphism was analyzed by genotyping, single-strand conformation polymorphism (SSCP) analysis, and allele-specific polymerase chain reaction (PCR). By PCR and direct sequencing, two new polymorphisms, C-1445G in the EGR promoter region and T216 (A>C) in the exon 3 of the TIEG1 gene were identified and analysed. Both polymorphisms as well as TNF- C-863A and C-857T, IL-1 C-889T, and IL-1 C-511T polymorphisms were analyzed by restriction enzyme digestion of PCR products. IL-1 3'UTR polymorphism was analyzed by SSCP. Among these eight polymorphisms studied, EGR C-1445G showed association with VUR development, whereas KLK1 -130 GN and TNF- C-863A polymorphisms with VUR progression. The fragments containing KLK1 -130 GN and EGR C-1445G polymorphisms were fused to a firefly luciferase reporter and transiently expressed in HEK-293 cells. Significant lower transcriptional activity was observed with -130 G12 K allele and EGR -1445 G allele.

    目 錄 I 中文摘要 IV 英文摘要 V 圖表次 VI 壹、緒論 一、膀胱輸尿管逆流 1 二、KLK1基因與其啟動子多型性 4 三、TIEG1/EGRα基因 6 四、TNF-基因與其啟動子多型性 7 五、IL-1、IL-1基因與其多型性 9 六、研究動機及目的 10 貳、研究材料與方法 一、樣品 12 二、基因組DNA的萃取 12 三、TIEG1/EGRα基因多型性的檢視 12 (一) 聚合酶鏈反應(PCR) 12 (二) 自洋菜膠中純化DNA片段 13 (三) DNA定序 13 (四) 多型性的檢視 13 四、KLK1、TIEG1/EGRα、TNF-、IL-1、IL-1基因的多型性分析 13 (一) 聚合酶鏈反應(PCR) 13 (二) 基因型分析(genotyping) 14 (三) 限制片段長度多型性(RFLP)分析 14 (四) 單股核酸構形多型性(SSCP)分析 15 (五) 直接定序(direct sequencing) 15 (六) 統計分析 15 五、啟動子片段的選殖(cloning) 16 (一) 聚合酶鏈反應(PCR)與DNA片段純化 16 (二) 接合反應(ligation) 16 (三) 轉形勝任細胞(competent cell)之製備 16 (四) 細菌的轉形作用(transformation) 17 (五) 質體DNA的小量置備及DNA定序 18 (六) 質體DNA的大量置備及純化 18 六、KLK1啟動子重組質體的構築 19 七、EGRα啟動子重組質體的構築 20 八、啟動子的功能性檢測 20 參、結果 一、KLK1基因啟動子-130GN多型性分析 22 二、KLK1基因-130GN多型性對啟動子功能的影響 22 三、TIEG1/EGRα基因多型性檢視與分析 23 四、EGRα基因C-1445G多型性對啟動子功能的影響 24 五、TNF-基因的多型性分析 25 六、IL-1基因的多型性分析 26 七、IL-1基因的多型性分析 27 肆、討論 28 伍、參考文獻 34

    1. Cotran, R.S., et al. Robbins pathologic basis of disease. 1989: W.B. Saunders Company.
    2. Dillon, M.J., Goonasekera, C.D. Reflux nephropathy. J Am Soc Nephrol 1998;9:2377-83.
    3. Kamil, E.S. Recent advances in the understanding and management of primary vesicoureteral reflux and reflux nephropathy. Curr Opin Nephrol Hypertens 2000;9:139-42.
    4. Devriendt, K., et al. Vesico-ureteral reflux: a genetic condition? Eur J Pediatr 1998;157:265-71.
    5. Eccles, M.R., et al. Unravelling the genetics of vesicoureteric reflux: a common familial disorder. Hum Mol Genet 1996;5:1425-9.
    6. Kaefer, M., et al. Sibling vesicoureteral reflux in multiple gestation births. Pediatrics 2000;105:800-4.
    7. Noe, H.N. The relationship of sibling reflux to index patient dysfunctional voiding. J Urol 1988;140:119-20.
    8. de Vargas, A., et al. A family study of vesicoureteric reflux. J Med Genet 1978;15:85-96.
    9. Jerkins, G.R., Noe, H.N. Familial vesicoureteral reflux: a prospective study. J Urol 1982;128:774-8.
    10. Andriole, V.T. The role of Tamm-Horsfall protein in the pathogenesis of reflux nephropathy and chronic pyelonephritis. Yale J Biol Med 1985;58:91-100.
    11. Chertin, B., et al. Upregulation of angiotensin II receptors in reflux nephropathy. J Pediatr Surg 2002;37:251-5.
    12. Clements, J.A. The molecular biology of the kallikreins and their roles in inflammation. In The Kinin System (Farmer, S., ed.), 1997: p. 71-79.
    13. Beaubien, G., et al. Gene structure and chromosomal localization of plasma kallikrein. Biochemistry 1991;30:1628-35.
    14. Riegman, P.H., et al. Characterization of the human kallikrein locus. Genomics 1992;14:6-11.
    15. Scicli, A.G., Carretero, O.A. Renal kallikrein-kinin system. Kidney Int 1986;29:120-30.
    16. Busse, R., Fleming, I. Molecular responses of endothelial tissue to kinins. Diabetes 1996;45:S8-13.
    17. Mahabeer, R., Bhoola, K.D. Kallikrein and kinin receptor genes. Pharmacol Ther 2000;88:77-89.
    18. Bhoola, K.D., et al. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 1992;44:1-80.
    19. Yu, H., et al. Association of the tissue kallikrein gene promoter with ESRD and hypertension. Kidney Int 2002;61:1030-9.
    20. Song, Q., et al. DNA polymorphisms in the 5'-flanking region of the human tissue kallikrein gene. Hum Genet 1997;99:727-34.
    21. Elliot, R., Nuzum, F.R. Urinary excretion of a depressor substance (kallikrein of Frey and Kraut) in arterial hypertension. Endocrinology 1934;18:462-74.
    22. Fautsch, M.P., et al. TGF beta-inducible early gene (TIEG) also codes for early growth response alpha (EGR alpha): evidence of multiple transcripts from alternate promoters. Genomics 1998;51:408-16.
    23. Subramaniam, M., et al. Identication of a novel TGF-beta regulated gene encoding a putative zinc finger protein in human osteoblasts. Nucleic Acids Res 1995;23:4907-12.
    24. Subramaniam, M., et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 1998;68:226-36.
    25. Blok, L.J., et al. Isolation of cDNAs that are differentially expressed between androgen-dependent and androgen-independent prostate carcinoma cells using differential display PCR. Prostate 1995;26:213-24.
    26. Cook, T., et al. Three conserved transcriptional repressor domains are a defining feature of the TIEG subfamily of Sp1-like zinc finger proteins. J Biol Chem 1999;274:29500-4.
    27. Tachibana, I., et al. Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 1997;99:2365-74.
    28. Subramaniam, M., et al. Tissue, cell type, and breast cancer stage-specific expression of a TGF-beta inducible early transcription factor gene. J Cell Biochem 1998;68:226-36.
    29. Chalaux, E., et al. A zinc-finger transcription factor induced by TGF-beta promotes apoptotic cell death in epithelial Mv1Lu cells. FEBS Lett 1999;457:478-82.
    30. Ribeiro, A., et al. The transforming growth factor beta(1)-inducible transcription factor TIEG1, mediates apoptosis through oxidative stress. Hepatology 1999;30:1490-7.
    31. Tachibana, I., et al. Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 1997;99:2365-74.
    32. Cheng, J., Grande, J.P. Transforming growth factor-beta signal transduction and progressive renal disease. Exp Biol Med 2002;227:943-56.
    33. Blok, L.J., et al. Characterization of an early growth response gene, which encodes a zinc finger transcription factor, potentially involved in cell cycle regulation. Mol Endocrinol 1995;9:1610-20.
    34. Carswell, E.A., et al. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 1975;72:3666-70.
    35. Beutler, B., Cerami, A. The biology of cachectin/TNF--a primary mediator of the host response. Annu Rev Immunol 1989;7:625-55.
    36. Black, R.A., et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997;385:729-33.
    37. Vassalli, P. The pathophysiology of tumor necrosis factors. Annu Rev Immunol 1992;10:411-52.
    38. Old, L.J. Tumor necrosis factor (TNF). Science 1985;230:630-2.
    39. Beutler, B. TNF, immunity and inflammatory disease: lessons of the past decade. J Investig Med 1995;43:227-35.
    40. Beutler, B.A., et al. Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol 1985;135:3972-7.
    41. Tracey, K.J., et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986;234:470-4.
    42. Higuchi, T., et al. Polymorphism of the 5'-flanking region of the human tumor necrosis factor (TNF)-alpha gene in Japanese. Tissue Antigens 1998;51:605-12.
    43. Skoog, T., et al. A common functional polymorphism (C-->A substitution at position -863) in the promoter region of the tumour necrosis factor-alpha (TNF-alpha) gene associated with reduced circulating levels of TNF-alpha. Hum Mol Genet 1999;8:1443-9.
    44. Bidwell, J., et al. Cytokine gene polymorphism in human disease: on-line databases. Genes Immun 1999;1:3-19.
    45. Kaluza, W., et al. Different transcriptional activity and in vitro TNF-alpha production in psoriasis patients carrying the TNF-alpha 238A promoter polymorphism. J Invest Dermatol 2000;114:1180-3.
    46. Kroeger, K.M., et al. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997;34:391-9.
    47. Wilson, A.G., et al. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA 1997;94:3195-9.
    48. Soga, Y., et al. Tumor necrosis factor-alpha gene (TNF-alpha) -1031/-863, -857 single-nucleotide polymorphisms (SNPs) are associated with severe adult periodontitis in Japanese. J Clin Periodontol 2003;30:524-31.
    49. Nothwang, H.G., et al. Molecular cloning of the interleukin-1 gene cluster: construction of an integrated YAC/PAC contig and a partial transcriptional map in the region of chromosome 2q13. Genomics 1997;41:370-8.
    50. Copeland, N.G., et al. Chromosomal location of murine and human IL-1 receptor genes. Genomics 1991;9:44-50.
    51. Dinarello, C.A. Proinflammatory cytokines. Chest 2000;118:503-8.
    52. Dinarello, C.A. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095-147.
    53. Colotta, F., et al. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 1993;261:472-5.
    54. Greenfeder, S.A., et al. Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity. J Biol Chem 1995;270:22460-6.
    55. McDowell, T.L., et al. A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1 alpha polymorphism. Arthritis Rheum 1995;38:221-8.
    56. Bailly, S., et al. Genetic polymorphism of human interleukin-1 alpha. Eur J Immunol 1993;23:1240-5.
    57. Bensen, J.T., et al. Association of an IL-1A 3'UTR polymorphism with end-stage renal disease and IL-1 alpha expression. Kidney Int 2003;63:1211-9.
    58. Hurme, M., Santtila, S. IL-1 receptor antagonist (IL-1Ra) plasma levels are co-ordinately regulated by both IL-1Ra and IL-1beta genes. Eur J Immunol 1998;28:2598-602.
    59. Combarros, O., et al. Interleukin-1A (-889) genetic polymorphism increases the risk of multiple system atrophy. Mov Disord 2003;18:1385-6.
    60. Hurme, M., et al. Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases. Ann Med 1998;30:469-73.
    61. Loughrey, B.V., et al. An interluekin 1B allele, which correlates with a high secretor phenotype, is associated with diabetic nephropathy. Cytokine 1998;10:984-8.
    62. Syrjanen, J., et al. Polymorphism of the cytokine genes and IgA nephropathy. Kidney Int 2002;61:1079-85.
    63. 劉國保. RAS系統和TFG-b1 的基因多型性與原發性VUR疾病發生和腎病進行的相關性研究. 國立師範大學生物學系碩士論文, 中華民國九十二年六月二十二日.
    64. Payami, H., et al. Relative predispositional effects (RPEs) of marker alleles with disease: HLA-DR alleles and Graves disease. Am J Hum Genet 1989;45:541-6.
    65. Reid, I.A., et al. The renin-angiotensin system. Annu Rev Physiol 1978;40:377-410.

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