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目的:观察EPA体外诱导FRH-0201细胞凋亡机理的初步研究。方法:在处于指数生长期的人肝胆管癌细胞株FRH-0201培养剂中添加二十碳五烯酸(EPA),采用流式细胞仪对FRH-0201细胞线粒体跨膜电位的影响、细胞色素c免疫组化染色及Western Bolt检测Casepase-9和Casepase-3的表达。结果:FRH-0201细胞线粒体Δψm下降随EPA剂量的增大而显著增多(P<0.05),EPA处理组细胞内棕色颗粒的强弱和阳性细胞的比例明显高于对照组,差异具有统计学意义(P<0.05,P<0.001),EPA不同浓度处理FRH-0201细胞48h后,Caspase-3和Caspase-9蛋白表达显著提高。结论:EPA可能通过降低线粒体膜电位,释放细胞色素C,激活Caspase-9,从而引发Caspase-3的级联反应,诱导FRH-0201细胞凋亡。
Objective: To observe the mechanism of EPA inducing apoptosis in FRH-0201 cells in vitro. Methods: Eicosapentaenoic acid (EPA) was added to human hepatocellular carcinoma line FRH-0201 in exponential growth phase. The effect of flow cytometry on the mitochondrial transmembrane potential of FRH-0201 cells was studied. Cytochrome c immunohistochemical staining and Western Bolt detection Casepase-9 and Casepase-3 expression. Results: The decrease of mitochondrial Δψm in FRH-0201 cells was significantly increased with the increase of EPA dosage (P <0.05). The intensity of brown granules and the percentage of positive cells in EPA-treated cells were significantly higher than those in control group (P <0.05, P <0.001). The expression of Caspase-3 and Caspase-9 protein in FRH-0201 cells treated with different concentrations of EPA for 48 h was significantly increased. Conclusion: EPA may induce caspase-3 cascade and induce apoptosis of FRH-0201 cells by decreasing mitochondrial membrane potential, releasing cytochrome C and activating Caspase-9.