华蟾素诱导人肝癌细胞株HepG2凋亡及其作用机制

为了观察华蟾素体外抑制肝癌细胞株HepG₂增殖、诱导细胞凋亡作用及其机制, 将不同浓度华蟾素作用于HepG₂细胞。采用MTT法观察华蟾素对HepG₂细胞的增殖抑制作用; 用Hoechst 33258染色法观察细胞凋亡的形态学改变; 以流式细胞术观察细胞的凋亡率和细胞周期的变化; 以实时荧光定量PCR技术 (real time-PCR) 和蛋白免疫印迹 (Western blotting) 法检测细胞凋亡相关因子Bcl-2、Bax及p53 mRNA水平和蛋白水平的表达情况。结果表明, 华蟾素对体外培养的肝癌HepG₂细胞有抑制增殖作用, 且具有剂量、时间相关性; 华蟾素可致HepG₂细胞阻滞于G₂/M期, 细胞凋亡率随药物浓度增加亦呈增长趋势; 细胞凋亡相关因子Bax及p53 mRNA和蛋白表达上调, Bcl-2的mRNA和蛋白表达下调。因此, 华蟾素可抑制肝癌细胞株HepG₂的增殖, 诱导肝癌HepG₂细胞凋亡, 其机制可能与调节凋亡相关因子Bcl-2、Bax及p53的表达有关。

Apoptosis-inducing effect of cinobufacini on human hepatoma cell line HepG2 and its mechanism of action

To investigate the apoptosis-inducing effect of cinobufacini (Huachansu) on human hepatoma cell line HepG₂ and its possible mechanism of action, HepG₂ cells were treated with different concentrations of   cinobufacini.  Cell proliferation was measured by methylthiazolyl tetrazolium (MTT) assay.  The morphological changes of apoptosis were observed by Hoechst 33258 staining.  Cell cycle distribution and apoptotic rate were evaluated by flow cytometry (FCM).  Quantitative real-time RT-PCR and Western blotting analysis were used to detect the mRNA and protein expressions of apoptosis related factors Bcl-2, Bax and p53.  The results indicated that cinobufacini could inhibit the proliferation of HepG₂ cells in a dose and time dependent manner.  Remarkable morphological changes of apoptosis including cytoplasmic and nuclear condensation and partition of cytoplasm were observed by Hoechst 33258 staining.  According to FCM analysis, HepG₂ cells were arrested in G₂/M phase and the apoptotic rate increased with the increase of the concentration of cinobufacini.  Both the mRNA and protein expressions of Bax and p53 were up-regulated while Bcl-2 expression down-regulated.  Thus,  cinobufacini could inhibit the proliferation and induce apoptosis of human hepatoma cell line HepG₂.    Furthermore, up regulation of Bax and p53 as well as down regulation of Bcl-2 expressions may be one of the important apoptotic inducing mechanisms.