【摘要】 目的:探讨羟基喜树碱(HCPT)诱导SMMC-7721细胞凋亡过程中凋亡诱导因子(AIF)不同时相的定位变化和DNA损伤的关系。方法:HCPT作用SMMC-7721细胞后在不同时间点收集细胞,用细胞组分分离试剂盒分离细胞质、线粒体与细胞核成分,用蛋白印迹法与共聚焦显微镜观察AIF的转位,用TUNEL法检测DNA损伤。结果:蛋白印迹结果显示线粒体中AIF在HCPT作用1h无明显变化,6h出现了明显转位,在12h与24h更明显;共聚焦结果也显示在HCPT作用6h后,AIF已从线粒体迁出,12h与24h后,大部分AIF已从线粒体迁至细胞核;TUNEL法检测结果表明伴随AIF的核迁移,DNA出现了明显的损伤。结论:HCPT作用SMMC-7721细胞后AIF的释放是凋亡的早期改变之一;伴随AIF的核转位,同时出现了DNA损伤。更多还原

【Abstract】 Objective:To investigate the relationship between transportation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and DNA damage at different time points during apoptosis of SMMC-7721 cells induced by hydroxycamptothecin (HCPT). Methods:Cells were harvested at different time points after HCPT treatment. The mitochondrium, cytosol, and nucleus were isolated with mitochondria isolation kit. These fractions were used for western blot. Transportation of AIF from mitochondrium to the nucleus at different time points was observed by confocal microscopy. At the same time, DNA damage was detected by TUNEL assay. Results:Western blot showed that transportation of AIF from mitochondrium to nucleus was not observed after treatment with HCPT for 1 h. However, AIF immunoreactivity was significantly increased in the nuclear fraction at 6, 12 and 24 h after HCPT treatment. Confocal microscopy demonstrated that AIF was released from mitochondrium at 6 h after HCPT treatment. The massive transportation of AIF to the nucleus occurred at 12 and 24 h after HCPT treatment. At the same time, DNA damage was observed by TUNEL assay. Conclusion:AIF release is one of the early biochemical changes of apoptosis of SMMC-7721 cells after HCPT treatment. The DNA damage occurrs in parallel with AIF translation to nucleus.更多还原