去甲斑蝥素对肝癌细胞增殖和凋亡的影响

Effect of norcantharidin on proliferation and apoptosis of hepatoma HepG2 cells

目的:研究去甲斑蝥素(norcantharidin,NCTD)对人肝癌HepG2细胞增殖、凋亡和survivin表达的影响。方法:MTT法检测NCTD对HepG2细胞增殖的影响,Hochest33258染色、AnnexinV/PI染色、DNA琼脂糖电泳检测NCTD对HepG2细胞凋亡的影响,Western blotting检测NCTD对HepG2细胞survivin蛋白表达的影响。结果:NCTD可显著抑制HepG2细胞的增殖,呈质量浓度(5、10、20和40μg/ml)依赖性,40μg/ml NCTD作用48h时HepG2细胞抑制率达(81.27±3.25)%。NCTD作用HepG2细胞24h后,荧光显微镜下可见HepG2细胞出现核固缩和核裂解现象;DNA琼脂糖电泳显示,基因组DNA呈现典型的凋亡梯状图形;流式细胞术结果显示,5、10、20和40μg/ml NCTD作用后,HepG2细胞的凋亡率分别为(7.33±0.25)%、(18.23±1.19)%、(32.5±2.30)%和(48.23±1.17)%。Western blotting结果显示,随着NCTD作用剂量的增加,HepG2细胞中survivin蛋白的表达逐渐减少。结论:NCTD能抑制HepG2细胞增殖并促进其凋亡,其凋亡的发生与下调survivin蛋白的表达有关。

Objective：To study the influence of norcantharidin （NCTD） on the proliferation,apoptosis and survivin expression of human hepatoma HepG2 cells. Methods：MTT assay was used to investigate the effect of NCTD on proliferation of HepG2 cells; Hochest 33258 staining,Annenxin V/PI staining,and agarose gel electrophoresis assay were used to examine the effect of NCTD on apoptosis of HepG2 cells. Western blotting analysis was used to examine the effect of NCTD on survivin expression in HepG2 cells. Results：Proliferation of HepG2 cells was markedly inhibited by NCTD in a dose-dependent manner （5,10,20,40 μg/ml）,with the inhibitory rate being （81.27±3.25）% after 40 μg/ml NCTD treatment for 48 h. After HepG2 cells were treated with NCTD for 24 h,fuorescence microscopy revealed that the cell nuclei were condensed and fragmented,and agarose gel electrophoresis of DNA showed a typical DNA ladder map of apoptosis. Flow cytometry results showed that the apoptosis rates of HepG2 cells were （7.33±0.25）%,（18.23±1.19）%,（32.5±2.30）%,and （48.23±1.17）% after 5,10,20,and 40 μg/ml NCTD treatments,respectively. Western blotting analysis results demonstrated that the survivin expression in HepG2 cells was significantly down-regulated with the increase of NCTD dosage. Conclusion：NCTD can inhibit proliferation and induce apoptosis of HepG2 cells,which is related to the down-regulation of survivin protein.