摘要
为探讨热休克预处理对过氧化氢所致C2C12肌原细胞凋亡和第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物从线粒体释放的影响 ,采用 0 .5mmol L过氧化氢作用于C2C12肌原细胞。Hoechst332 5 8荧光染色 ,观察细胞形态学改变并计算凋亡百分率 ,抽提DNA作琼脂糖电泳 ,以确定细胞凋亡情况。采用天冬氨酸特异性半胱氨酸蛋白酶活性定量检测试剂盒及蛋白质印迹观察天冬氨酸特异性半胱氨酸蛋白酶 3,天冬氨酸特异性半胱氨酸蛋白酶 9的活化情况。采用免疫荧光及细胞成分分离后蛋白质印迹检测第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物从线粒体的释放。结果发现 ,过氧化氢处理 1~ 2h ,第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物逐渐从线粒体释放入胞浆 ;处理 4h天冬氨酸特异性半胱氨酸蛋白酶 3,天冬氨酸特异性半胱氨酸蛋白酶 9活性升高 ,12h达高峰 ;处理 2 4h细胞明显出现凋亡 ,凋亡百分率明显升高。热休克预处理可诱导C2C12肌原细胞中热休克蛋白 90 ,热休克蛋白 70及αB 晶状体蛋白的表达增高 ;抑制第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物释放、天冬氨酸特异性半胱氨酸蛋白酶 3,天冬氨酸特异性半胱氨酸蛋白酶 9的活化及细胞凋亡的发生。结果提示 ,线粒体信号通路在?
Aim To explore the effect of heat shock pretreatment on hydrogen peroxide (H 2O 2)-induced apoptosis and the second mitochondria-derived activator of caspases (Samc) release from mitochondria in C2C12 myogenic cells. Methods After heat shock pretreatment (42℃ 1 h, recover 12 h), C2C12 myogenic cells were exposed to H 2O 2 (0.5 mmol/L)for 6 h, 12 h, 24 h,and 36 h respectively. The apoptotic morphological changes and percentage of apoptotic nuclei of C2C12 myogenic cells were analyzed. Caspase-3, Caspase-9 activities were assayed by caspase colorimetric assay kit and Western-blot. Inducible heat shock proteins were detected using Western-blot analysis. The release of Smac from mitochondria to cytoplasm was observed by Western-blot and immunofluorescence analysis. Results ①After treated with H 2O 2 (0.5 mmol/L), a marked increase of mitochondrial Smac release, activation of caspase-9, caspase-3 and apoptosis were observed respectively in C2C12 myogenic cells.②Heat shock pretreatment induced expression of HSP70, HSP90,αB-crystallin and inhibited H 2O 2-mediated Smac release from mitochondria, and inhibited the activation of caspase-9, caspase-3 and subsequent apoptosis. Conclusions Heat shock pretreatment could protect C2C12 myogenic cells against H 2O 2-induced apoptosis, and its mechanism might involve heat shock proteins expression and inhibition of Samc release from mitochondria.
出处
《中国动脉硬化杂志》
CAS
CSCD
2004年第2期125-130,共6页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金 ( 3 0 0 0 0 0 69和 3 0 2 70 5 3 3 )
教育部博士点专项基金 ( 2 0 0 2 0 5 3 3 0 3 2 )
国家 973重点项目 (G2 0 0 0 0 5 690 8)资助