目的研究三氧化二砷(As2O3)诱导人肝癌细胞系Bel-7402细胞凋亡及与线粒体跨膜电位的关系。方法应用不同浓度的As2O3作用于肝癌细胞后,观察As2O3对肝癌细胞生长状态的影响;通过噻唑蓝(MTT)比色法观察其对Bel-7402细胞增殖的影响;利用共聚焦显微镜及流式细胞术观察经Annexin V-FITC/PI双染后的细胞早期凋亡;通过共聚焦显微镜及流式细胞仪检测线粒体跨膜电位(MMP)的改变情况;并通过底物染色法反映Caspase-3的活性。结果不同浓度的As2O3作用于肝癌细胞后有明显的时间和剂量依赖性;经Annexin V-FITC/PI双染后,可观察到Bel-7402细胞的早期凋亡现象,2μmol/L As2O3作用24h细胞凋亡率为9.89%,作用48h细胞凋亡率为48.53%,而4μmol/L As2O3作用24h细胞凋亡率为18.27%,作用48h细胞凋亡率为67.52%;经As2O3药物作用后,细胞线粒体膜电位水平下降,且与药物作用时间和药物浓度有关(P〈0.05);同时Caspase-3活性被激活。结论As2O3可明显抑制肝癌细胞Bel-7402的生长,并使细胞线粒体膜电位下降,诱导肝癌细胞凋亡。
Objective To investigate the relationship between Bel-7402 cell apoptosis induced by arsenic trioxide (As2O3) and cell mitochondrial membrane potential. Methods After treatment with As2O3 of different concentrations (0.5-8 μmol/L), the effect of As2O3 on the growth of liver cancer cell line Bel-7402 was observed. The inhibitory effect of As2O3 on the cell proliferation was investigated with 2-(4,5-dimethyl-thizazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) method and its inducibility of cell apoptosis detected by confocal microscopy and flow cytometry after dying with Annexin V-FITC/PI. The mitochondrial membrane potential was determined by confocal microscopy and flow cytometry and the activation of caspase-3 was assayed by substrate color reaction. Results The effect of As2O3 on liver cancer cell.line Bel-7402 was dependent on the time and concentration obviously. The apoptotic cells were detected by confocal microscopy and flow cytometry after dying with Annexin V-FITC/PI. The mitochondrial membrane potential decreased in a dose-time dependent manner and the caspase-3 was activated. Conclusions As2O3 can significantly inhibit the growth of liver cancer cell line Bel-7402, induce its apoptosis and decrease the mitochondrial membrane potential.