蟾蜍灵对EAhy926细胞增殖、凋亡及Caspase3,Bcl-2表达的影响

Effects of bufalin on Proliferation,apoptosis and expression of Caspase3,Bcl-2 in EA hy926A cells

【目的】探讨蟾蜍灵对血管内皮细胞EAhy926的增殖抑制作用及其机制。【方法】体外培养血管内皮细胞EAhy926,用不同浓度蟾蜍灵进行干预。通过四甲基偶氮唑盐（MTT）法检测细胞增殖抑制率;用倒置光显微镜观察细胞形态变化;用Hoechst33342/PI双荧光染色,荧光显微镜观察细胞核形态变化;以流式细胞术检测细胞凋亡的比率;应用半定量逆转录PCR检测EAhy926细胞中Caspase3和Bcl-2的mRNA表达。【结果】蟾蜍灵对EAhy926细胞生长具有抑制作用;且在0.0625~1μmol/L范围内呈时间、剂量依赖性;荧光显微镜下可见0.1μmol/L的蟾蜍灵作用24 h可诱导EAhy926细胞凋亡,作用48 h可引起细胞部分坏死;流式细胞术检测出典型的＂凋亡峰＂,凋亡率8.2%~36.5%,且呈时间依赖性。半定量RT-PCR显示Caspase3 mRNA表达较对照组明显增强,Bcl-2 mRNA表达较对照组明显减弱,且二者均呈剂量依赖性。【结论】蟾蜍灵抑制EAhy926细胞增殖、诱导其凋亡,其机制可能与上调Caspase3和下调Bcl-2基因表达有关。

【Objective】 To investigate the inhibitory effects of bufalin on proliferation of EAhy926 vascular endothelial cell and involved mechanisms.【Methods】Vascular endothelial cell EAhy926 were cultivated in vitro and treated with various concentrations of bufalin.The growth inhibition rate of cells was measured by methyl-tetrazolium（MTT） method;The morphological changes were detected by light microscope;The feature of cell death was studied by Hoechst33342/PI staining;Cell apoptosis rate were detected by flow cytometry（FCM） and the mRNA expression of Caspase3 and Bcl-2 were measured by reverse transcription PCR（RT-PCR）.【Results】 The growth of EAhy926 cell was inhibited by bufalin with concentration and time dependent manners from 0.0625~1 μmol/L.Enhancement of proportion of apoptotic cell were observed by fluorescence microscope and bufalin in high concentration induced cell necrosis partially.Apoptosis peak was detected by FCM,and the apoptotic rate was between 8.2%~36.5% with time dependent manner.The result of RT-PCR showed that the mRNA expression of Caspase3 was higher and the mRNA expression of Bcl-2 was lower than the control group with concentration dependent manner.【Conclusion】The effect of bufalin inhibiting EAhy926 cell and inducing cell apoptosis may be related to upregulating the expression of Caspase3 and downregulating the expression of Bcl-2.