肾脏固有细胞表型转化中α-sma基因甲基化状态的研究

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1、中图分类号：R318.04；R319；R363.2+1硕士学位论文肾脏固有细胞表型转化中α-SMA基因甲基化状态的研究院（系）、所基础医学院研究生姓名荣智利学科、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目录肾脏固有细胞表型转化中α-SMA基因甲基化状态的研究„111.1中文摘要„„„„„„„„„„„„„„„„„„„„„31.2英文摘要„„„„„„„„„„„„„„„„„„„„„51.3前言„„„„„„„„„„„„„„„„„„„„„„„81.4材料与方法„„„„„„„„„„„„„„„„„„„„121

2、.5结果„„„„„„„„„„„„„„„„„„„„„„„271.6讨论„„„„„„„„„„„„„„„„„„„„„„„341.7结论„„„„„„„„„„„„„„„„„„„„„„„461.8参考文献„„„„„„„„„„„„„„„„„„„„„471.9英汉缩略词对照表„„„„„„„„„„„„„„„„„532致谢„„„„„„„„„„„„„„„„„„„„„„„553巨噬细胞在肾小管上皮-间质细胞转分化中的作用（综述）„„„„„„„„„„„„„„„„„„„„„„„„„564在读期间发表论文复印件2肾脏固有细胞表型转化中α-S

3、MA基因甲基化状态的研究摘要目的：应用焦磷酸测序技术对肾脏固有细胞表型转化标志性蛋白α-SMA的基因转录起始点附近序列的甲基化状态进行定位、定量检测，分析比较肾小球系膜细胞（HBZY-1）及肾间质成纤维细胞（NRK-49F）在表型转化前后α-SMA基因的甲基化图示及数量的差异，从表观遗传学水平阐述肾脏固有细胞发生表型转化的可能机制，为肾脏纤维化的早期防治及寻找新的治疗靶标提供新的可行性研究思路。方法：（1）分别对HBZY-1及NRK-49F进行体外培养，同步化后两株细胞均分为无血清培养的正常对照组和重组人TGF

4、-β1（10ug/L，48h）诱导的诱导组各两组。各组细胞分别进行形态学观察、细胞爬片及培养上清液收集，细胞爬片进行免疫细胞化学染色观察细胞胞浆内α-SMA蛋白表达情况，ELISA检测培养上清液中Ⅰ型胶原含量，分别比较两株细胞各组实验结果，判定诱导条件诱导细胞发生表型转化的结果。（2）根据实验前部分验证结果，采用同等条件诱导细胞表型转化，收集各组新鲜细胞，提取全基因组DNA，应用焦磷酸测序技术检测α-SMA基因转录起始点附近甲基化状态，比较两株细胞各组甲基化情况。结果：（1）诱导后的两株细胞均发生形态学改变，表

5、现为细胞胞体增大，胞浆丰富，胞核增大，HBZY-1由梭形或不规则星形变为多角形或树枝形，扩张胞突连接成片，NRK-49F由长梭形变为短梭形或多角形，部分可见胞内空泡。（2）细胞免疫化学检测显示，正常HBZY-1胞浆内仅有少量α-SMA蛋白呈弱阳性表达，诱导后胞浆内α-SMA蛋白呈强阳性高表达（P＜0.05）；正常NRK-49F胞浆内无α-SMA蛋白表达，诱导后胞浆α-SMA蛋白出现3并呈强阳性高表达（P＜0.05）。（3）ELISA检测显示，正常HBZY-1及NRK-49F的培养上清液中均含有Ⅰ型胶原，诱导后两

6、株细胞培养上清液中Ⅰ型胶原含量均明显增多（P＜0.05）。（4）正常HBZY-1及NRK-49F中α-SMA基因呈现不同程度甲基化，HBZY-1甲基化频率在20%-50%之间呈低甲基化状态，NRK-49F甲基化频率＞50%呈高甲基化状态，诱导后两株细胞中α-SMA基因甲基化图示及数量均无变化。结论：（1）TGF-β1可诱导HBZY-1及NRK-49F发生细胞表型转化，转变为高表达α-SMA蛋白、能分泌大量细胞外基质的肌成纤维细胞，在肾脏纤维化形成中的发挥重要作用。（2）α-SMA基因转录起始点附近甲基化状态在肾

7、脏固有细胞表型转化中作用尚不能完全明确，是否通过表观遗传修饰中DNA甲基化状态的改变促进肾脏纤维化的发展有待进一步研究。关键词：焦磷酸测序；肾脏固有细胞；表型转化；肾脏纤维化4Studyonthemethylationstatusofα-SMAinthephenotypic-changeofrenalresidentialcellsAbstract:Objective:Thesequenceoftranscriptioninitiationregionofα-SMAthatissymbolicproteinof

8、phenotypic-changedofrenalresidentialcellswaslocationandrationdetectedthemethylationstatusbypyrosequencing.Thentoanalyzethedifferenceofillustrationandamoutofmethylationthatbelongtoα-SMAgeneinthef