HSP70抑制H_2O_2所致C2C12肌原细胞Smac从线粒体的释放及细胞凋亡  

HSP70 inhibits Smac release from the mitochondria and protects against H_2O_2-induced apoptosis in C2C12 myogenic cells

作　　者：蒋碧梅[1] 肖卫民[1] 石永忠[1] 刘梅冬[1] 肖献忠[1] 

机构地区：[1]中南大学湘雅医学院病理生理学教研室,长沙410078

出　　处：《中南大学学报（医学版）》2005年第1期32-37,共6页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金项目(30000069;30270533);国家973重点项目(G2000056908);教育部博士点专项基金(20020533032)

摘　　要：目的:阐明HSP70对H2O2所致C2C12肌原细胞凋亡的影响及其作用机制。方法:采用基因瞬间转 染技术使细胞HSP70或/和Smac高表达,采用Hoechst33258染色观察H2O2(0.5mmol/L)处理转基因C2C12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带。采用 caspase活性定量检测试剂盒及蛋白质印迹分析caspase 9和caspase 3的活化。利用免疫荧光分析HSP70对H2O2 所致Smac从线粒体释放的影响。结果:HSP70对H2O2及Smac所致C2C12肌原细胞凋亡具有明显的抑制作用,凋 亡核百分率明显降低,DNA电泳未出现明显“梯状”条带;caspase 9和caspase 3的活化明显受到抑制;并进一步发 现HSP70可抑制H2O2所致C2C12肌原细胞Smac从线粒体释放。结论:HSP70可通过抑制Smac从线粒体释放来Objective To observe whether HSP70 could protect against H 2O 2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria. Methods HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33258 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 the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence. Results H 2O 2 (0.5mmol/L) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H 2O 2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H 2O 2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H 2O 2. Conclusion HSP70 inhibits Smac release from the mitochondria and protects against H 2O 2-induced apoptosis in C2C12 myogenic cells.

关 键 词：HSP70 SMAC 细胞凋亡 H2O2 C2C12肌原细胞 

分 类 号：R361.1[医药卫生—病理学]