过氧化氢诱导C_2C_(12)细胞凋亡的机制初探

Preliminary Investigation on the Mechanism of C_2C_(12) Cell Apoptosis induced by Hydrogen Peroxide

【目的】探讨氧化应激诱导小鼠胚胎肌原细胞株C2 C12 细胞凋亡的分子机制。【方法】采用 0 .5mmol/L过氧化氢 (hydrogenperoxide,H2 O2 )作用于C2 C12 细胞 ;通过Hoechst荧光染色检测C2 C12 细胞凋亡 ,Caspase活性定量分析及Western blot检测Caspase 3, 8, 9活化情况 ,Western blot及间接免疫荧光检测细胞色素C在细胞内的分布情况。【结果】本实验发现H2 O2 能明显诱导C2 C12 细胞凋亡 ,同时Caspase 3, 8, 9被激活 ,而细胞色素C从线粒体释放入胞浆。【结论】氧化应激可通过同时激活线粒体通路与死亡受体通路导致C2 C12 细胞凋亡 。

ObjectiveTo investigate the molecular mechanism that apoptosis of mouse embryonic myogenic cell line C 2C 12 cells was induced by oxidative stress.Apoptosis of mouse C 2C 12 cells was induced by exposure to 0.5 mmol/L hydrogen peroxide (H 2O 2) for different durations, and their apoptotic changes were detected by Hoechst 33258 fluorescence staining. The activities of caspase -3, -8, -9 were examined by caspase colorimetric assay kit and Western-blotting. The intracellular distribution of cytochrome C and its release from mitochondria were observed by indirect immunofluorescence and Western-blotting.Exposure to 0.5 mmol/L H 2O 2 for 24 hours markedly induced C 2C 12 cell apoptosis as shown by Hoechst 33258 fluorescence staining. The activities of caspase -3, -8, -9 significantly increased after 4 hours of H 2O 2 treatment, and reached their peaks at 8～12 hour. The release of cytochrome C from mitochondria to cytoplasm was detected after exposure to H 2O 2 for 1～2 hours.[Conclusions]Oxidative stress can induce apoptosis of C 2C 12 cells through simultaneous activation of mitochondria and death receptor signal pathways. This result provides new information for clinical prophylaxis and treatment of apoptosis related cardiovascular diseases.