低浓度乙醇对人肝癌HepG2细胞的杀伤作用

目的探讨低浓度乙醇对人肝癌Hep G2细胞的杀伤作用,及其作用机制。方法以体外培养的人肝癌Hep G2细胞为研究对象,MTT法检测细胞毒性,吖啶橙(AO)、溴化乙锭(EB)染色观察细胞凋亡形态,2’,7’-二氢二氯荧光黄双乙酸钠(DCFH-DA)法检测Hep G2细胞内活性氧族(ROS)水平,罗丹明123(Rh123)染色检测线粒体膜电位变化,免疫荧光法检测Caspase-9表达,流式细胞术检测细胞凋亡率。结果 MTT显示,低浓度乙醇呈时间依赖性和剂量依赖性抑制Hep G2细胞增殖,1.0、1.6mol/L乙醇作用6h后,细胞内ROS、Caspase-9荧光强度均显著高于对照组(P0.01,n=20),Rh123荧光强度显著低于对照组(P0.01,n=20);高内涵活细胞成像系统可见细胞核固缩呈圆珠状凋亡特征;流式细胞术显示,1.0mol/L乙醇作用6h可诱导27.92%Hep G2细胞凋亡,5.4%细胞坏死;1.6mol/L乙醇作用6h可诱导31.22%Hep G2细胞凋亡,15.1%Hep G2细胞坏死。结论低浓度乙醇可通过升高细胞内ROS水平,降低线粒体膜电位,上调Caspase-9表达,通过线粒体途径诱导Hep G2细胞凋亡。

Inhibitory effect of low concentration ethanol on HepG2 cells

Objective To study the effect of low concentration ethanol on apoptosis of human hepatocellular carcinoma HepG2 cells, and to explore the mechanism. Methods MTT assay was used to evaluate the cell inhibition rate. The cellular morphous was detected with AO/EB staining, and the ROS was detected with DCFH-DA staining, the mitochondrial membrane potential（ΔΨm）was detected with Rh123 staining,and the expression of Caspase-9 was detected by immunofluorescence technic. Apoptosis of HepG2 cells was quantified by flow cytometry using Annexin V/PI stain. Results Proliferation of HepG2 cells was inhibited by low concentration ethanol in a dosage and time dependent manner. Under HCS, some HepG2 cells underwent typical apoptotic morphologic change and the expression of ROS and Caspase-9 was increased. The mitochondrial membrane potential（ΔΨm）was decreased. Flow cytometry indicated 27.92% HepG2 cells were induced apoptosis and 5.4% HepG2 cells were necrotic after 6 hours incubation with 1.0mol/L ethanol, and 31.22% HepG2 cells were induced apoptosis and 15.1% HepG2 cells were necrotic after 6 hours incubation with 1.6mol/L ethanol. Conclusion The low concentration ethanol induces the apoptosis of HepG2 cells by the mitochondrial pathway. The mechanism is concerned with increasing ROS, decreasing the mitochondrial membrane potential and upgrading Caspase-9. 