自噬参与促进AFB1致肝细胞DNA损伤的修复进程  

Autophagy involves in accelerating the repair of AFB1-induced DNA damage in hepatocytes

作　　者：夏斌[1] 廖昆[1] 张玉静[1] 邱杨[2] 骆冰[2] 庄群瑛[2] 殷花[1] 何承勇[2] 林育纯[1,2] 林忠宁[1,2] 

机构地区：[1]中山大学公共卫生学院,广东广州510080 [2]厦门大学公共卫生学院,福建厦门361102

出　　处：《热带医学杂志》2014年第10期1263-1266,F0004,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(81172705;81072334);广东省自然科学基金(S2011020002769);福建省自然科学基金(2014J01372)

摘　　要：目的探讨黄曲霉毒素B1(AFB1)肝毒性修复进程中细胞自噬的作用。方法以经50μmol/L AFB1预处理24 h的永生化人正常肝L02细胞建立模型,检测AFB1处理撤除不同时间点的细胞DNA双链损伤指标γH2AX蛋白和自噬标志物LC3-Ⅱ蛋白的表达水平;采用免疫荧光法检测AFB1撤除后细胞中γH2AX蛋白荧光焦点和自噬体形成情况;在AFB1撤除后联合应用自噬抑制剂3-MA进行干预实验,检测细胞内γH2AX蛋白的表达水平。结果L02细胞中γH2AX蛋白的动态表达在AFB1处理撤除12 h后达到最高,并在24 h后逐渐降低;自噬蛋白LC3-Ⅱ和自噬体的形成在AFB1处理撤除后逐渐增强;3-MA可明显减缓AFB1撤处理后细胞内γH2AX蛋白的降低进程。结论细胞自噬参与了AFB1诱导肝细胞毒性损伤的修复进程,与促进DNA损伤的修复有关。Objective To investigate the role of autophagy plays in the repair process of aflatoxin B1（AFB1）-induced hepatotoxicity. Methods The immortal normal human hepatic L02 cell line was used in vitro experiments. L02 cells were exposed to 50 μmol / L AFB1 for 24 h, followed by a culture in the absence of test chemical for serial time-courses, and the expression of γH2AX, a hallmark of double-strand DNA breaks（DSBs）, and LC3-Ⅱ, an autophagic marker, were detected by Western blotting. In addition, L02 cells were subjected to γH2AX dots or autophagosome detection by immunofluorescence assay post-exposure of AFB1. L02 cells were treated with AFB1 for 24 h followed by withdrawal of exposure extensively, and incubated in the presence or absence of 3-MA for 24 h and 48 h and the cell lysates were prepared to determine γH2AX expression. Results The induced γH2AX protein peaked at 12 h and remained detectable at various time post-exposure to AFB1, while it gradually returned to background after 24 h. Autophagy detection showed a continuously increase both in LC3-II expression and autophagosome formation after AFB1 withdrawal. The reduction of γH2AX expression was gradually proceeding in the 3-MA negative population, whereas the effect was totally inhibited by 3-MA treatment. Conclusion The results indicate that the cellular autophagy involves in the repair process of AFB1-induced hepatotoxicity and relates to the rescue of DNA damage after AFB1 withdrawal.

关 键 词：黄曲霉毒素B1 自噬 肝细胞 DNA损伤修复 γH2AX 

分 类 号：R114[医药卫生—卫生毒理学]