补体片段C3a对体外培养足细胞表型的影响  

Effect of complement fragment C3a on the phenotype of mice podocytes in vitro

作　　者：邓金秀[1] 陈财铭[1] 郑露莉 崔炯[1] 许艳芳[1] 邹臻寰[1] 李镇洲[1] 万建新[1] Deng Jinxiu;Chen Caiming;Zheng Luli;Cui Jiong;Xu Yanfang;Zou Zhenhuan;Li Zhenzhou;Wan Jianxin(Department of Nephrology, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Chin)

机构地区：[1]福建医科大学附属第一医院肾内科,福州350005

出　　处：《中华肾脏病杂志》2018年第2期106-114,共9页Chinese Journal of Nephrology

基　　金：福建省自然科学基金(2015J01390);福建省卫生与计划生育委员会青年科研课题(2015-1-55)

摘　　要：目的探讨补体片段C3a对小鼠足细胞表型转化的影响。方法外源性C3a干预分化成熟的足细胞，用RT．PCR、Western印迹、免疫化学和免疫荧光方法检测足细胞synaptopodin、podocin、nephrin、CD2相关蛋白（CD2AP）、成纤维细胞特异性蛋白1（FSP-1）、a-平滑肌肌动蛋白（仪。SMA）mRNA和蛋白的表达。经整合素连接激酶（ILK）siRNA预处理后的足细胞再加入外源性C3a干预，用Western印迹检测nephrin和a-SMA蛋白的表达，Western印迹和免疫化学方法检测Snail和d—actinin4蛋白的表达，细胞划痕实验观察足细胞体外迁移能力。结果免疫化学和免疫荧光检测结果显示，分化成熟足细胞表达synaptopodin、podoein、nephrin、CD2AP，0．1μmol／LC3a刺激24h后其表达减少；分化成熟足细胞表达少量FSP．1、a-SMA，0．1μmol／LC3a刺激24h后其表达增加。与对照组相比，随着C3a浓度的增加synaptopodin、podocin、CD2AP和nephrin的mRNA表达减少（均P〈0．05），FSP-1和a-SMA的mRNA表达增加（均P〈0．05）。Western印迹结果显示，与对照组相比，随着C3a浓度增加，足细胞synaptopodin、podocin、nephfin、CD2AP的蛋白表达减少（均P〈0．05），FSP-1、a-SMA的蛋白表达增加（均P〈0．05）。与C3a直接干预组比较，足细胞经ILKsiRNA预处理后再加入外源性C3a，nephfin蛋白表达增加（P〈0．05），d．SMA蛋白表达减少（P〈0．05）；同时，a-actinin4和Snail的蛋白表达减弱，足细胞迁移能力下降。结论补体片段C3a可诱导体外培养的小鼠足细胞向间充质细胞转化，ILK信号通路参与细胞表型的转化。Objective To investigate the effect of complement C3a on mouse podocytes phenotype transformation. Methods Purified C3a recombinant protein was used to stimulate mature mouse podocytes. The expression of the mature podocyte markers synaptopodin, podoein, nephrin, CD2- associated protein （CD2AP） and the mesenehymal cell markers fibroblast specific protein 1 （FSP-1）, a- smooth muscle actin （a- SMA） were detected by RT- PCR, Western blotting, immunochemistry and immunofluorescence, respectively. Some podocytes were transfeeted with integrin-linked kinase （ILK） siRNA before the administration of C3a, the expression of nephrin and a- SMA were aecessed by Western blotting, and the expression of Snail and ct-aetinin 4 were accessed by Western blotting and immunochemical method. The migration ability of podocytes was observed by scratch test. ResultsImmunocytochemistry and immunofluorescence analysis showed that synaptopodin, podocin, nephrin, CD2AP were highly expressed by mature mouse podocytes. The expression of these podocyte markers could be markedly inhibited after 24 h of C3a （0.1 Ixmol/L） treatment, and accompanied by the induction of mesenchymal markers FSP-1 and a-SMA. Compared with control group, the mRNA levels of synaptopodin, podocin, CD2AP and nephrin were significantly repressed by the administration of C3a in a dose- dependent manner, whereas the transcription of FSP- 1 and a- SMA were remarkably up- regulated by C3a treatment （P 〈 0.05, respectively）. Western blotting analysis also confirmed the decrease of synaptopodin, podocin, nephrin and CD2AP protein and the increase of FSP-1 and a-SMA protein were closely depend on the C3a concentration （P 〈 0.05, respectively）. To further assess the downstream of C3a, some podocytes were transfected with ILK siRNA before the administration of C3a. Compared with C3a group, the protein levels of nephrin and a-SMA were significantly changed by the administration of ILK siRNA （P 〈 0.05, respectively）. The expression of a-actinin

关 键 词：补体C3a 足细胞 上皮-间充质转分化 整合素连接激酶信号通路 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]