雷帕霉素通过激活自噬减轻亚溶量补体诱导的足细胞黏附损伤  

Rapamycin reduces podocyte adhesion damage caused by sublytic C5b-9 via autophagy activation

作　　者：吕倩影[1] 周建华[1] 陈瑜[1] 杨凤杰[1] 蒲金赟 张瑜[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《中华肾脏病杂志》2014年第10期751-756,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81100514,30772360）;教育部博士点基金（新教师类）（20110142120017）;华中科技大学自主创新研究基金（2013QN192）

摘　　要：目的 观察雷帕霉素对亚溶量补体C5b-9（sublytic C5b-9,sC5b-9）所致足细胞黏附损伤的影响,并探讨细胞自噬在其中的作用.方法 建立sC5b-9攻击足细胞体外模型,采用透射电镜观察足细胞自噬泡形成,罗丹明标记鬼笔环肽（rhodamine phalloidin）染色后激光共聚焦显微镜下观察细胞骨架蛋白F-actin的分布及细胞形态,细胞黏附实验评价足细胞黏附能力,Western印迹检测微管相关蛋白1轻链3（microtubule-associated protein l light chain 3,LC3）Ⅰ型（LC3-Ⅰ）和Ⅱ型（LC3-Ⅱ）的表达,流式细胞术检测整联蛋白α3的表达.结果 （1）成功建立sC5b-9攻击足细胞体外模型,将细胞溶破率≤5％定义为亚溶量攻击;（2）透射电镜和自噬标志LC3-Ⅱ的检测均显示造模后48 h足细胞的自噬水平明显增强;（3）3-甲基腺嘌呤抑制自噬可明显加剧sC5b-9所致的足细胞黏附能力下降及细胞骨架病变,但对整联蛋白α3的表达无明显影响;（4）雷帕霉素干预可显著改善sC5b-9所致的足细胞黏附能力受损及细胞骨架破坏;（5）雷帕霉素明显增强sC5b-9所诱导的足细胞自噬.结论 雷帕霉素可通过增强自噬的途径直接改善亚溶量补体所诱导的足细胞黏附损伤.Objective To determine the effect of rapamycin on sublytic C5b-9-induced podocyte adhesion damage,and whether autophagy is involved in this progression.Methods Sublytic complement C5b-9 stimulation was used in vitro.Autophagosomes were viewed using electron microscopy.Western blotting was used to measure the change of autophagy-related markers.Attachment assay was used to assess the adhesion of podocyte.Confocal microscopy was used to explore the expression patterns of cytoskeletal protein F-actin.Flow cytometry was used to measure the level of adhesion-associated protein integrin α3.Results （1） For ensuring sublytic complement injury,the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis （defined as ＞ 5％ LDH release）.（2） Sublytic C5b-9 promoted autophagy in podocyte in vitro.The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated autophagosomes and enhanced expression of LC3-lⅡ.（3） Inhibition of autophagy by 3-methyadenine enhanced the effect of sublytic C5b-9-induced podocyte injury,including serious cytoskeleton damage and markedly reduced adhesion of podocyte.（4） Rapamycin treatment significantly improved the above lesions.（5） Rapamycin enhanced autophagy induced by sublytic C5b-9 in podocyte.Conclusions In summary,rapamycin can improve sublytic CSb-9-induced podocyte adhesion damage by appropriate autophagy activation.These findings provide important information for the development of appropriate protocols for the application of mTOR （mammalian target of rapamycin） inhibitors in podocytopathy.

关 键 词：自噬 足细胞 西罗莫司 亚溶量补体 

分 类 号：R96[医药卫生—药理学]