ecadherin和αsma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、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„„„„„„„„„„„„„„„„„„„„111.5结果„„„„„„„„„„„„„„„„„„„„„„„241.6讨论„„„„„„„„„„„„„„„„„„„„„„„301.7结论„„„„„„„„„„„„„„„„„„„„„„„

2、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

3、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：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48

4、h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

5、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)。4）经焦磷酸测序的定位、定量检测两组细胞E

6、-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E-cadh

7、erin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:Pyrosequencingt

8、echniquewasbeenusedfordetectionoftherelationshipofE-cadherinandalphaSMAgenepromoterregionmethylationstatusandgeneexpressionintheEMTprocessofNRKcells,wecanmakein-depthunderstandingoftheroleofepigeneticintheEMTprocessofNRKcells,Itwillhelp