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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:233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

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