CAJ | 학술논문

目的：通过亚溶量补体C5b-9（sC5b-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sC5b-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。 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sC5b-9所致的足细胞形态异常。 sC5b-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sC5b-9所诱导的足细胞凋亡。结论：sC5b-9可直接诱导足细胞自噬活动增强，而自噬则在sC5b-9介导的足细胞损伤过程中发挥着保护作用。
목적：통과아용량보체C5b-9（sC5b-9）공격족세포적리체모형，탐토자서재면역개도족세포손상과정중적작용。방법：건립sC5b-9공격족세포체외모형，채용단단광선시알（MDC）염색표기자서포，서씨-길모살염색광경하관찰세포형태，면역형광검측nephrin적표체급분포，Western blotting 검측LC3-II화LC3-I적표체，MTT법검측세포존활솔，Annexin V／PI쌍염법류식세포술검측세포조망。결과：성공건립sC5b-9공격족세포체외모형，장세포용파솔≤5％정의위아용량공격。 MDC염색화자서표지LC3-II적검측균현시조모후48 h족세포적자서활동명현활약。억제자서가명현가중sC5b-9소치적족세포형태이상。 sC5b-9공격가사족세포nephrin적표체량명현감소，병차이상분포，이억제자서가진일보하조nephrin적표체。억제자서가촉진sC5b-9소유도적족세포조망。결론：sC5b-9가직접유도족세포자서활동증강，이자서칙재sC5b-9개도적족세포손상과정중발휘착보호작용。
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 .