Staurosporine诱导乳鼠心肌细胞凋亡与Bcl-2/Bax的关系

Relationship between Bcl-2/Bax and staurosporine-induced rat cardiomyocyte apoptosis

目的探讨staurosporine(STS)诱导心肌细胞凋亡过程中,凋亡调节蛋白Bcl-2和Bax的变化及意义。方法以STS诱导原代培养的SD乳鼠心肌细胞,检测半胱天冬蛋白酶(caspase)-3的活性及用Hoechst-33258荧光染色观察细胞凋亡。用免疫印迹法(Western blot)检测Bcl-2和Bax表达的变化。用免疫荧光共聚焦显微镜和免疫印迹法观察Bax在细胞内的分布。结果以4μmol/L STS处理心肌细胞,随着处理时间的延长,细胞中caspase-3的活性逐渐增加,与对照组比较,8 h达到峰值(P<0.01)。Hoechst-33258荧光染色显示,多数细胞核呈现核分叶。STS诱导心肌细胞凋亡过程中,Bcl-2和Bax的水平与对照组比较无显著变化。正常细胞内的Bax均匀分布于细胞质中,在STS处理的细胞中,Bax明显聚集于线粒体上,呈现明显的线粒体的转位。STS诱导心肌细胞凋亡过程中,胞质片段Bax的水平明显下降,而线粒体片段Bax的水平明显升高,进一步证实STS诱导细胞凋亡过程中伴有明显的Bax线粒体转位。结论STS诱导细胞凋亡中,伴有明显的Bax由胞质向线粒体的转位。

AIM To explore the changes of Bcl-2/Bax and significance in staurosporine （STS）induced cardiomyocyte apoptosis. METHODS Primary cultured neonatal rat cardiomyocytes were exposed to STS to induce apoptosis. Cell apoptosis was determined by cysteine aspartate specific proteinase （caspase）-3 activity and Hoechst 33258 staining. Bcl-2/Bax expression was measured by Western blot and Bax translocation was assessed by double immunofluorescence labeling as well as Western blot. RESULTS Exposure of cardiomyocytes to STS （4μmol/L） resulted in an increase of caspase-3 activity in a time-dependent manner with a peak at 8 h. Hoechst 33258 fluorescence staining showed many segmented and irregularly shaped nuclei and STS induction did not alter Bcl-2/Bax levels. In normal cardiomyocytes, a diffuse localization of Bax was primarily found in the cytoplasm. In contrast, exposure of cells to STS resulted in Bax translocation from cytoplasm to mitochondria. Western blot also demonstrated that an increase in mitochondrial Bax level was accompanied by a concomitant decrease in cytosolic Bax level, which provided some direct evidence that a significant increased Bax translocation existed in the STS-induced cell apoptosis. CONCLUSION STS induces a significant pro-apoptotic Bax translocation to mitochondria in cardiomyocyte apoptosis, a mechanism that may be involved in STS-induced cell apoptosis.