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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。
Objective To investigate the changes and significance of apoptotic regulatory proteins (Bcl-2 and Bax) in cardiomyocytes apoptosis induced by staurosporine (STS). Methods Primary cultured neonatal rat cardiomyocytes were stained with STS and the activity of caspase-3 was detected. Hoechst-33258 staining was used to observe the apoptosis. The changes of Bcl-2 and Bax expression were detected by Western blot. Immunofluorescence confocal microscopy and Western blotting were used to observe the distribution of Bax in the cells. Results Cardiomyocytes were treated with STS at 4μmol / L, and the activity of caspase-3 increased gradually with the increase of treatment time, reaching the peak at 8 h (P <0.01). Hoechst-33258 fluorescence staining showed that most of the nuclei showed nuclear lobes. STS-induced cardiomyocyte apoptosis, Bcl-2 and Bax levels compared with the control group no significant change. Normal cells within the Bax evenly distributed in the cytoplasm, STS-treated cells, Bax was significantly gathered in the mitochondria, showing obvious translocation of mitochondria. During the process of STS-induced cardiomyocyte apoptosis, the level of cytoplasmic Bax decreased significantly, while the level of mitochondrial Bax was significantly increased, which further confirmed that Bax mitochondrial translocation accompanied with STS-induced apoptosis. Conclusion STS induces apoptosis with obvious translocation of Bax from cytoplasm to mitochondria.