摘要
该研究利用蛋白质免疫印迹法(Western blot)、稳定表达m Cherry-EGFP-LC3B(microtubuleassociated proteins 1A/1B light chain 3B,LC3)自噬双标荧光细胞、mt-Keima荧光探针活细胞扫描技术在人宫颈癌细胞He La中检测了多种自噬相关标志物,探究了阿司咪唑(astemizole,AST)对He La细胞自噬的影响。结果表明,阿司咪唑在He La细胞中引起自噬标志蛋白质LC3-II与SQSTM1/p62的显著累积,并存在剂量和时间依赖性效应;阿司咪唑导致He La细胞存活率显著降低;用自噬抑制剂Bafilomycin A1(Baf-A1)阻断自噬流(autophagic flux)后,再加入阿司咪唑不能促进LC3-II进一步累积;阿司咪唑处理细胞后自噬体与自噬溶酶体的数量均显著增加;此外,阿司咪唑显著抑制了线粒体氧化磷酸化解偶联剂羰基氰化物间氯苯腙(carbonyl cyanide 3-chlorophenylhydrazone,CCCP)导致的线粒体标志蛋白质TOMM20(translocase of outer mitochondrial membrane 20)的降解,其线粒体自噬水平显著低于对照组。以上结果证明了阿司咪唑有抑制自噬流的作用,导致损伤的线粒体不能被顺利降解,并且提示该作用可能是通过抑制溶酶体功能实现的。
To investigate the effects of Astemizole（AST） on regulating autophagy in He La cells, we used Western blot, m Cherry-EGFP tandem fluorescent-tagged LC3B（microtubule-associated proteins 1 A/1 B light chain 3 B, LC3） stable expression system, mt-Keima based living cell fluorescence imaging method in this study. The results of Western blot showed that AST resulted in dose-and time-dependent accumulation of LC3-II and SQSTM1/p62 in He La cells. And AST could not facilitate the further accumulation of LC3-II in combination with Bafilomycin A1（Baf-A1）, a blocker of autophagic flux. MTS assay presented that AST promoted cell death. The m Cherry-EGFP-LC3B fluorescence system indicated that He La cells exposed to AST displayed greatly accumulation of autophagosomes as well as autolysosomes. Furthermore, the generic mitochondrial uncoupling agent CCCP（carbonyl cyanide 3-chlorophenylhydrazone） could induce mitochondrial autophagy, or mitophagy, which accompanied by the rapid degradation of mitochondrial outer membrane marker TOMM20（translocase of outer mitochondrial membrane 20）. Western blot showed that AST inhibited CCCP-induced TOMM20 degradation as chloroquine（CQ） and Baf-A1 did. In addition, the mitochondrial autophagy was obviously reduced in cells treated with AST detected by mt-Keima. These results implied that AST could significantly inhibit autophagic flux and the clearance of damaged mitochondria in He La cells.
作者
赵菲
徐广
陈佳意
李婷
韩秋影
张学敏
潘欣
Zhao Fei;Xu Guang;Chen Jiayi;Li Ting;Han Qiuying;Zhang Xuemin;Pan Xin(National Center of Biomedical Analysis, Beijing 100850, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2018年第4期522-532,共11页
Chinese Journal of Cell Biology
基金
国家重大科学研究计划(批准号:2014CB910603)
北京市科技专项(批准号:Z151100000315085)资助的课题~~
