e-cadherin和α-sma基因的表观调控在肾间质纤维化中作用的研究

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1、中图分类号：R363；R365硕士学位论文E-cadherin和α-SMA基因的表观调控在肾间质纤维化中作用的研究院（系）、所基础医学院研究生姓名江燕学科、专业病理学与病理生理学导师姓名马跃荣（教授）二Ο一二年四月1目录E-cadherin和α-SMA基因的表观调控在肾间质纤维化中作用1的研究„„„„„„„11.1中文摘要„„„„„„„„„„„„„„„„„„„„„31.2英文摘要„„„„„„„„„„„„„„„„„„„„„51.3前言„„„„„„„„„„„„„„„„„„„„„„„81.4材料与方法„„„„„„„„„„„„„„„„„„„

2、„111.5结果„„„„„„„„„„„„„„„„„„„„„„„241.6讨论„„„„„„„„„„„„„„„„„„„„„„„301.7结论„„„„„„„„„„„„„„„„„„„„„„„371.8参考文献„„„„„„„„„„„„„„„„„„„„„381.9英汉缩略词对照表„„„„„„„„„„„„„„„„„422致谢„„„„„„„„„„„„„„„„„„„„„„„43表观遗传学应用于慢性肾病研究的进展（综3述）„„„„„„„„„„„„„„„„„„„„„„„„„442E-cadherin和α-SMA基因的表观调控在肾间质纤维化中作用的研究摘要

3、目的：采用焦磷酸测序技术对大鼠肾小管上皮细胞间质转分化（Epithelial-mesenchymaltransition,EMT）过程中E-钙黏附蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-smoothmuscleactin，α-SMA）基因启动子区甲基化状态进行定位、定量检测，分别比较肾小管上皮细胞在表型转化前后E-cadherin基因以及α-SMA基因的甲基化状态及数量差异，以期揭示表观遗传学在肾小管上皮细胞EMT过程中的作用，有助于进一步阐明肾间质纤维化发生发展的机制，进而为临床上慢性肾病的早期防治提供新的方向。方法

4、：将体外培养的NRK细胞株随机分为两组：①正常对照组；②TGF-β1干预组（10ng/ml）。两组细胞干预培养48h后，观察NRK细胞形态学的变化，细胞免疫化学染色半定量检测贴壁细胞中E-cadherin以及α-SMA蛋白表达量，酶联免疫吸附实验（ELISAs）检测两组细胞上清液中I型胶原蛋白的浓度，提取收集细胞中的基因组DNA，应用焦磷酸测序技术分别对两组细胞中E-cadherin和α-SMA基因启动子区甲基化水平进行检测。分析比较NRK细胞诱导前后两种基因特定区域甲基化状态变化。结果：1）培养48h后，正常对照组的NRK细胞形态

5、仍为椭圆形的铺路砖样，而TGF-β1干预组中NRK细胞形态发生显著改变，由椭圆形的铺路砖样的上皮细胞形态转变为长梭形的成纤维细胞样形态；2）3培养48h后，正常组NRK细胞胞浆中上皮标志性蛋白E-cadherin呈高表达，TGF-β1干预组细胞胞浆内无E-cadherin表达(P<0.001)；正常组NRK细胞胞浆中无间质源性标志性蛋白α-SMA表达，TGF-β1干预组细胞胞浆内α-SMA的表达显著增加(P<0.001)。3）与正常对照组相比，培养48h后，TGF-β1干预组细胞培养上清液中的I型胶原蛋白含量显著增加(P<0.001

6、)。4）经焦磷酸测序的定位、定量检测两组细胞E-cadherin基因目的片段上的8个CpG位点以及α-SMA基因目的片段上的6个CpG位点的甲基化状态，可发现，TGF-β1干预组细胞中E-cadherin基因甲基化频率高于正常空白组（P<0.001）；TGF-β1干预组细胞中α-SMA基因甲基化频率低于正常对照组（P<0.001）。结论：1）经10ng/ml的TGF-β1干预NRK细胞48h后可迅速建立理想的肾小管上皮细胞EMT和肾间质纤维化体外模型；2）E-cadherin基因启动子区CpG岛高甲基化状态导致基因失活，使上皮标志物

7、E-cadherin蛋白表达减少参与肾小管上皮细胞EMT过程；3）α-SMA启动子区CpG岛低甲基化状态导致基因激活，使间充质细胞标志物α-SMA蛋白表达增加参与肾小管上皮细胞EMT过程。关键词：焦磷酸测序技术；甲基化；上皮-间质转分化；肾间质纤维化4EffectofE-cadherinandalphaSMAgeneepigeneticregulationintheprocessofrenalinterstitialfibrosiseffectAbstract:Objective:Pyrosequencingtechniquewas

8、beenusedfordetectionoftherelationshipofE-cadherinandalphaSMAgenepromoterregionmethylationstatusandgeneexpressioninthe