肼致心肌成纤维细胞凋亡作用机制的研究

目的观察肼作用于体外培养心肌成纤维细胞的凋亡情况并探讨其机制。方法将新生大鼠心肌成纤维细胞培养标本分为对照组和肼处理组。用2mmol／L浓度的肼处理细胞72h后Hoeehst33258染色，荧光显微镜下观察凋亡细胞核的变化；膜蛋白V／磺化丙啶双染流式细胞仪检测细胞凋亡率和坏死率；Rodamine123荧光染色流式细胞仪检测细胞内线粒体膜电位的变化；Western blot检测凋亡相关蛋白Bax／Bob2和Caspase-3表达的变化。结果肼处理后的细胞与对照组相比，出现明显的细胞凋亡的核浓缩、核聚集形态；细胞凋亡率和坏死率与对照组比较差异均有显著性；细胞内线粒体膜电位降低；Bax表达增高、Bcl-2表达降低、Caspase-3表达增高。结论肼可引起心肌成纤维细胞凋亡，并可能通过线粒体通路即肼损伤线粒体引起Bcl-2表达降低，Bax表达增加致使线粒体膜通透性转换孔开放，膜电位降低，细胞色素C释放入胞浆，激活Caspase-3，引起细胞凋亡。

Experimental study on mechanism of the apoptosis of cardiac fibroblasts induced by hydrazine

Objective To observe the apoptosis effect of hydrazine on cardiac fibroblasts(CFB) cultured in vitro, so as to investigate its mechanism. Methods Specimens of cardiac fibroblasts cul- tured from heart tissue of neonatal rats were divided into control group and hydrazine group. The cul- tured fibroblasts of hydrazine group were treated with 2 mmol/L hydrazine for 72 h. The cell nucleus changes of apoptosis were examined by fluorescence microscope using Hoechst33258 stain. The rates of apoptosis and necrosis of cardiac fibroblasts stained by Annexin V-FITC/Propidiun Iodide Apoptosis Detect Kit I were detected by flowcytometry. The mitochondria membrane potential of cells stained by rhodamine123 was assayed by flowcytometry. The expression of Caspase-3, Bax, Bcl-2 were detected by Western-blot. Results The nucleus of apoptosis cardiac fibroblasts showed nucleus aggregation phenomenon. As compared with control group, the rates of apoptosis and necrosis in hydrazine treated group were significantly higher, while the mitochondria membrane potential of cells was significantly lower, the expression of Caspase-3 and the Bax expression were increased, while the Bcl-2 expression decreased. Conclusions Hydrazine can induce CFB apoptosis. The possible mechanism is that hy- drazine leads injury of mitochondria membrane and decrease of the mitochondria membrane potential, which in turn leads opening of transition pore,and release of cytochrome C into cytosol interacting with apoptotic protease-activating factor-1(Apaf-1), ATP/dATP, and pro-Caspase-9 forming apoptosome, and then activating pro-Caspase-3, results in CFB apoptosis.