摘要
利用Western杂交检测自噬标记物LC3Ⅱ和p62的表达是评价细胞自噬活性的常用方法。为了比较作用于不同靶点的细胞自噬调节剂对LC3Ⅱ和p62表达的影响,分别利用以mTOR依赖或非依赖方式活化细胞自噬的雷帕霉素或海藻糖、抑制自噬起始的3-甲基腺嘌呤、抑制自噬小体与溶酶体融合的巴弗洛霉素A1以及抑制溶酶体酶活性的E64d和pepstatin A处理HEK293细胞,通过Western杂交检测LC3Ⅱ和p62的表达。结果显示,雷帕霉素增强LC3I向LC3Ⅱ转化,促进p62降解,而海藻糖仅引起LC3Ⅱ表达的上调;阻断细胞自噬过程各个阶段均导致LC3Ⅱ和p62堆积,并呈现出剂量和时间依赖性。这些结果提示尽管LC3Ⅱ和p62均为自噬标记物,但不同的调节剂对其表达的调控存在差异。
Western blotting of autophagic markers LC3Ⅱ and p62 are widely used for estimating autophagic activity. To compare the regulation of various autophagy modulators on LC3Ⅱ and p62,HEK293 cells were treated separately with m TOR-dependent autophagy activator rapamycin or-independent autophagy activators trehalose,and autophagy inhibitors including 3-methyladenine（ 3-MA）,bafilomycin A1 or E64 d and pepstatin A that inhibited the initiation of autophagy, the fusion of autophagosome and lysosome, and the activities of lysosomal enzymes accordingly,and then LC3Ⅱ and p62 levels were assessed. Western blot results demonstrated that rapamycin enhanced the conversion of LC3 I to LC3Ⅱ,promoted the degradation of p62 simultaneously,while trehalose merely increased the expression of LC3Ⅱ with no influence on the p62 level. Moreover, inhibition of autophagy commonly led to accumulation of LC3Ⅱ as well as blockage of p62 degradation in a concentration-and timedependent manner. These results indicate that obvious differences exist in the regulation of LC3Ⅱ and p62 by various modulators although both are autophagic markers.
作者
魏砚明
任晋宏
栾智华
王永辉
WEI Yanming 1 ,REN Jinhong 1,LUAN Zhihua 2,WANG Yonghui 2(1 College of Pharmaceutical and Food Engineering; 2 Experimental Center,Shanxi University of Chinese Medicine,Jinzhong 030619,Chin)
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2018年第3期341-347,共7页
Journal of China Pharmaceutical University
基金
山西省卫生计生委科研课题资助项目(No.201601114)
山西中医药大学博士启动基金资助项目(No.2015BK16)~~
