活细胞中丝裂霉素诱导肺腺癌细胞凋亡机理的分析

An Analysis of MMC-induced ASTC-a-1 Apoptosis in Living Cells

为了分析丝裂霉素(mitomycin C,MMC)引起的肺腺癌细胞(ASTC-a-1)凋亡,实验通过CCK-8试剂盒检测不同浓度MMC对ASTC-a-1活性的影响,选择10μg/mL的MMC处理ASTC-a-1。利用Hochest 33258染色观察MMC引起的ASTC-a-1凋亡,发现MMC可引起细胞缩小,核浓缩。为了进一步研究MMC引起凋亡的途径,通过脂质体转染pSCAT3质粒,利用光漂白技术和光谱技术观察Caspase-3是否活化;通过转染Bax和Ds-Red质粒观察Bax在凋亡过程中的位置变化及与线粒体的关系。结果显示:MMC可以诱导ASTC-a-1细胞内Caspase-3活化,并使Bax向线粒体转移聚集。在活细胞中证实Caspase-3和Bax参与了MMC引起的ASTC-a-1凋亡。

To investigate mitomycin C （MMC）-induced apoptosis of ASTC-a-1, the cells were treated with MMC at differcut couceutration and their activity was assessed by cell counting kit （ CCK-8）. Based on results of CCK-8, cells were treated with 10 μg/ml, of MMC. Hochcst 33258 has been used to verifiy the MMC-induced cell apoptosis and the results indicated that MMC induced cell and nuclear shrinked. For advanced investigation of the apoptosis pathway induced by MMC, human lung adenocarcinoma cells （ ASTC-a-1 ） was transfected with SCAT3 plasmid, a fluorescence resonance energy transfer （FRET） plasmid. Photobleaching and emission spectrum techniques have been used to monitor the activation of Caspase-3 in living cells. ASTC-a-1 was transfected with plasmid Bax and Ds-Red to monitor the translocation of Bax into mitochondria during apoptosis induced by MMC. Our results demonstrate that both Caspase-3 activation and Bax are involved in the MMC-induced apoptosis.