Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢 (H2 O2 )所致C2 C12 肌原细胞凋亡中的作用 ,采用Hoechst 3 3 2 58染色 ,观察H2 O2 (0 5mmol/L)处理C2 C12 肌原细胞不同时间后 ,细胞核形态学改变并计算凋亡核百分率 ,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带 ,利用细胞成分分离后蛋白质印迹分析H2 O2 是否导致Smac/DIABLO从线粒体释放 ,采用Caspase检测试剂盒及蛋白质印迹分析Caspase 3和Caspase 9的活化 ,转染Smac/DIABLO基因 ,观察Smac/DIABLO过表达对H2 O2 所致的C2 C12 肌原细胞凋亡的影响 .结果表明 :H2 O2 处理 1h后 ,Smac/DIABLO从C2 C12 肌原细胞线粒体释放入胞浆 ,2h更明显 ;H2 O2 处理 4h后 ,Caspase 3和Caspase 9活化 ,12h达高峰 ;H2 O2 处理 2 4h后 ,C2 C12 肌原细胞显示特征性的凋亡形态改变 ,凋亡核百分率明显升高 ,DNA电泳出现明显“梯状”条带 .与单纯过氧化氢损伤组相比 ,Smac/DIABLO高表达的C2 C12 肌原细胞经过氧化氢损伤组的Caspase 3和Caspase 9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显 .结果表明 ,H2 O2 可导致Smac/DIABLO从C2 C12 肌原细胞线粒体释放 ,促进Caspase 9和Caspase 3的活化而促进细胞凋亡的发生

Role of Smac/DIABLO in H2O2-induced Apoptosis in C2C12 Myogenic Cells

In order to explore the role of Smac/DIABLO in H 2O 2-induced apoptosis of C 2C 12 myogenic cells, Hoechst 33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe DNA fragmentation. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blotting. Activities of Caspase-3, Caspase-9 were a ssayed with Caspase Colorimetric Assay Kit and Western blotting. Full length Smac/DIABLO gene was transiently transfected in C 2C 12 myogenic cells by lipofectamine and then protein levels of Smac/DIABLO were analysed by Western blotting. The results showed that: (1)H 2O 2 (0 5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondrial to cytoplasm 1 h after treatment, activation of Caspase-3, Caspase-9 4 h after treatment and specific morphological changes of apoptosis 24 h after treatment; (2)Smac/DIABLO overexpression significantly enhanced H 2O 2 induced apoptosis in C 2C 12 myogenic cells as shown by specific DNA ladder pattern in agarose gel electrophoresis, increase of percentage of apoptotic nuclei and marked activation of Caspase-3, Caspase-9 . These data suggested that H 2O 2 could result in a poptosis of C 2C 12 myogenic cells,and that release of Smac/DIABLO from mitochondrial to cytoplasm and the subsequent activation of Caspase-9 and Caspase-3 played important roles in H 2O 2-induced apoptosis in C 2C 12 myogenic cells.