摘要
目的:通过亚溶量补体C5b-9(s C5b-9)攻击足细胞的离体模型,探讨自噬在免疫介导足细胞损伤过程中的作用。方法:建立s C5b-9攻击足细胞体外模型,采用单丹磺酰尸胺(MDC)染色标记自噬泡,瑞氏-吉姆萨染色光镜下观察细胞形态,免疫荧光检测nephrin的表达及分布,Western blotting检测LC3-II和LC3-I的表达,MTT法检测细胞存活率,Annexin V/PI双染法流式细胞术检测细胞凋亡。结果:成功建立s C5b-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。MDC染色和自噬标志LC3-II的检测均显示造模后48 h足细胞的自噬活动明显活跃。抑制自噬可明显加重s C5b-9所致的足细胞形态异常。s C5b-9攻击可使足细胞nephrin的表达量明显减少,并且异常分布,而抑制自噬可进一步下调nephrin的表达。抑制自噬可促进s C5b-9所诱导的足细胞凋亡。结论:s C5b-9可直接诱导足细胞自噬活动增强,而自噬则在s C5b-9介导的足细胞损伤过程中发挥着保护作用。
AIM：In podocytes , autophagy occurs at a high basal level and dysregulated autophagy is associa -ted with a variety of podocytopathies .This paper is to investigate the role of autophagy in sublytic C 5b-9-induced podocyte injury.METHODS： Sublytic complement C5b-9 stimulation was used as an in vitro model.Autophagosomes were con-firmed using monodansylcadaverine （MDC） staining.Immunoblotting was used to measure the change of autophagy-related markers.Cellular morphological changes were observed by Wright-Giemsa staining.Immunofluorescence staining and con-focal microscopy were used to detect the expression and distribution of nephrin .The cell viability was assessed by methylth-iazol tetrazolium （MTT） assay.The cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/PI staining.RE-SULTS：For ensuring sublytic complement injury , the maximal amounts of anti-podocyte antiserum and 160 ×-diluted nor-mal human serum were used without inducing cell lysis （defined as 〉5%LDH release）.Sublytic C5b-9 promoted autoph-agy of podocytes in vitro.The proautophagic effect of sublytic C 5b-9 manifested in the form of accumulated MDC-labeled vesicles and enhanced the expression of LC 3-Ⅱ.Autophagy inhibitor 3-methyladenosine （3-MA） promoted sublytic C5b-9-induced podocyte morphological abnormalities .Compared with the sublytic C5b-9-injured podocytes, 3-MA exposure further decreased the expression of nephrin .3-MA enhanced sublytic C5b-9-induced podocyte apoptosis .CONCLUSION： Sub-lytic C5b-9 attack induces autophagy , which may play a protective role against complement-mediated podocyte injury .
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第1期59-63,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81100514)
教育部博士点基金(新教师类)(No.20110142120017)
华中科技大学自主创新研究基金(No.2013QN192)
