摘要
目的 研究缺氧对心肌细胞天冬氨酸特异性的半胱氨酸蛋白酶 (caspase)的激活效应。方法 培养的心肌细胞分别缺氧 0、3、6、12、2 4h后 ,Hoechst 3 3 3 42染色法检测细胞凋亡 ,MTT法检测细胞存活率 ,Westernblot、RT PCR方法分别检测细胞线粒体与胞浆中细胞色素c(Cytc)蛋白的变化以及caspase 9, 3mRNA表达的改变。结果 缺氧 2 4h心肌细胞出现典型的凋亡 ,细胞存活率显著降低 ;缺氧 3h线粒体Cytc未见显著变化 ,而胞浆中Cytc开始增多 ,缺氧 2 4h线粒体中已不能检测到Cytc ,胞浆中维持在峰值水平。缺氧可上调心肌细胞caspase 9, 3基因表达 ,两者表达变化趋势一致 ,缺氧 3h即开始明显增高 ,在观察的 2 4h内持续处于高表达状态。结论 缺氧可致心肌细胞线粒体Cytc大量释放至胞浆中 ,cas pase 9基因表达增高 ,并进一步导致caspase 3激活 ,从而证明缺氧可诱导心肌细胞中线粒体依赖的caspases途径激活 ,从而导致细胞凋亡。
Objective To explore the effects of hypoxia on the activation of caspases in cardiomyocytes. Methods At 0, 3, 6, 12, and 24 h after hypoxia, cell apoptosis and viability were determined with Hoechst 33342 staining and MTT method, respectively. Expressions of caspase-3, -9 mRNA and release of mitochondrial cytochrome c in primary culture of cardiomyocytes were determined by RT-PCR and Western blotting, respectively. Results Typical cell apoptosis was induced in cardiomyocytes at 24 h after hypoxia and cell viability decreased significantly. Elevation of Cyt c in cytosol was in accordance with the decline in mitochondrial Cyt c content. Significant increase in Cyt c in cytosol appeared at 12 h after hypoxia and peaked at 24 h while Cyt c in mitochondria could not be detected at 24 h after hypoxia. Hypoxia could up-regulate the caspase-3, -9 mRNA expressions at 3 h after exposure. Up-regulation of caspase-3, -9 mRNA expressions was maintained during 24 h hypoxic insult. Conclusion Hypoxia can induce mitochodrial Cyt c release into cytosol and up-regulate caspase-3, -9 mRNA expressions. These results demonstrate that hypoxia can induce mitochondrium-dependent caspase-3 activation in cardiomyocytes, resulting in cell apoptosis.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第3期185-188,共4页
Journal of Third Military Medical University
基金
国家重点基础研究发展规划资助项目 ("973"项目 ) (G19990 54 2 0 2 )~~
