蛋白酶体抑制剂Epoxomicin对视网膜色素上皮细胞自噬-溶酶体途径的影响  

Study on Influence of Autophagy-lysosome Pathway in ARPE-19 Cells by an Ubiquitin-proteasome Pathway Inhibitor Epoxomicin

作　　者：詹姣[1] 何洁儿[1] 商福[1] 吴明星[1] 张新愉[1] 

机构地区：[1]中山大学中山眼科中心//眼科学国家重点实验室,广东广州510060

出　　处：《中山大学学报（医学科学版）》2016年第4期508-513,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：卫生部部属(管)医院2013-2015年度国家自然科学青年基金项目(81200670)

摘　　要：【目的】研究抑制泛素-蛋白酶体途径(UPP)对自噬-溶酶体途径(ALP)的影响及相关作用机制。【方法】人视网膜色素上皮细胞系ARPE-19共分为3组,分别为DMEM空白对照组、DMEM+DMSO阴性对照组、DMEM+Epoxomicin实验组。应用蛋白免疫印迹法和细胞免疫荧光法检测泛素化蛋白的表达,观察UPP功能的改变情况;观察LC3,LAMP1、LAMP2的表达,评价自噬作用和溶酶体的功能变化情况;观察HDAC6,p62的表达,初步探索UPP抑制对ALP改变的分子机制。【结果】采用蛋白酶体抑制剂Epoxomicin抑制泛素-蛋白酶体途径后,细胞内泛素化蛋白质聚集物增多;自噬溶酶体途径被激活,自噬标志物LC3表达增高,溶酶体膜相关蛋白LAMP1、LAMP2蛋白水平表达亦增高;抑制UPP后HDAC6和p62表达升高,可能参与ALP的激活过程。【结论】泛素-蛋白酶体途径抑制后,自噬溶酶体途径被激活且参与蛋白质聚集物的降解。[Objective] To investigate the influence and mechanism upon inhibition of ubiquitin-proteasome pathway on autophagy-lysosome pathway. [Methods] Human retinal pigment epithelial cell line ARPE-19 cells were divided into three groups according to the culture medium： DMEM group, DMEM ＋DMSO group, DMEM ＋Epoxomicin group. The expression levels of ubiquitinated proteins were analyzed by western blot and immunofluorescence to determine the UPP activity. The activity of the ALP was determined by LC3- II , LAMP1 and LAMP2 expression, which was utilized to assess the functions of autophagy and lysosome. Meanwhile, the expression of HDAC6 and p62 were evaluated to elucidate the mechanism of the influence on ALP upon UPP inhibition. [Results] Inhibition of the UPP by epoxomicin resulted in aggregates of ubiquitinated proteins and protein aggregates marker -/-tubulin.The ALP was activated with the elevated level of LC3, LAMP1 and LAMP2. Levels of HDAC6 and p62 increased upon UPP inhibition, suggesting they may be involved in the activation of ALP when UPP was inhibited. [ Conclusions ] The ALP was activated and played a role in the degradation of protein aggregates as a consequence of UPP inhibition.

关 键 词：泛素 蛋白酶体 自噬 溶酶体 蛋白质聚集物 

分 类 号：R77[医药卫生—眼科]