CAJ | 학술논문

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.
목적 관찰뢰파매소대아용량보체C5b-9(sublytic C5b-9,sC5b-9)소치족세포점부손상적영향,병탐토세포자서재기중적작용.방법 건립sC5b-9공격족세포체외모형,채용투사전경관찰족세포자서포형성,라단명표기귀필배태(rhodamine phalloidin)염색후격광공취초현미경하관찰세포골가단백F-actin적분포급세포형태,세포점부실험평개족세포점부능력,Western인적검측미관상관단백1경련3(microtubule-associated protein l light chain 3,LC3)Ⅰ형(LC3-Ⅰ)화Ⅱ형(LC3-Ⅱ)적표체,류식세포술검측정련단백α3적표체.결과 (1)성공건립sC5b-9공격족세포체외모형,장세포용파솔≤5％정의위아용량공격;(2)투사전경화자서표지LC3-Ⅱ적검측균현시조모후48 h족세포적자서수평명현증강;(3)3-갑기선표령억제자서가명현가극sC5b-9소치적족세포점부능력하강급세포골가병변,단대정련단백α3적표체무명현영향;(4)뢰파매소간예가현저개선sC5b-9소치적족세포점부능력수손급세포골가파배;(5)뢰파매소명현증강sC5b-9소유도적족세포자서.결론 뢰파매소가통과증강자서적도경직접개선아용량보체소유도적족세포점부손상.
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.