中华眼镜蛇毒组分C对人肝癌细胞株BEL-7402细胞凋亡及端粒酶活性的影响

目的 :研究中华眼镜蛇毒组分C对人肝癌细胞株BEL 74 0 2细胞凋亡和端粒酶活性的影响 ,以探讨其抗肿瘤的作用机制。方法 :用不同浓度的中华眼镜蛇毒组分C分别处理人肝癌细胞株BEL 74 0 2 ,采用MTT法、流式细胞仪法、TRAP -ELISA、透射电镜等观察其对肝癌细胞的细胞毒、细胞凋亡、细胞周期及端粒酶活性的影响。结果 :中华眼镜蛇毒组分C对人肝癌细胞株BEL 74 0 2有明显的抑制作用 ,且呈时间依赖性及剂量依赖性。用流式细胞仪分析发现中华眼镜蛇毒组分C可诱导BEL 74 0 2肝癌细胞发生凋亡 ,肝癌细胞明显阻滞于G0 /G1 期 ,在G1 峰前出现明显的亚二倍体凋亡峰 ,且其凋亡率随蛇毒浓度的升高而呈上升的趋势。用TRAP ELISA法同步测得蛇毒作用BEL 74 0 2肝癌细胞 8h后的端粒酶活性值明显下降 ,其抑制作用与剂量呈正相关 (r =0 .96 4 ,P <0 0 5 )。在电镜下 ,可观察到肝癌细胞出现明显的凋亡特征性改变 :细胞膜完整、核染色质固缩 ,核碎裂、凋亡小体形成。结论 :中华眼镜蛇毒组分C可显著抑制人肝癌细胞株 (BEL 74 0 2 )的增殖 ,在诱导肿瘤细胞发生凋亡的同时也下调瘤细胞的端粒酶活性 ,这可能是FC抗肿瘤的重要机制之一。

The Effect of Telomerase Activity And Cell Apoptosis on Human Hepatocarcinoma Cell Line (BEL-7402) By Fraction C from Naja Naja Atra Venom

Objective:To investigate the anti cancer mechanism of Fraction C on human hepatocarcinoma cell line (BEL 7402) by observing toxic effect on cell apoptosis and the inhibition of telomerase activity in vitro.Methods:The cell activity and morphologic changes were studied after treated with different concentrations. MTT and Flow Cytometry (FCM) were applied in detecting the cytotoxic effect,dose dependency and cell apoptosis rate of FC on human hepatocarcinoma cell line (BEL 7402),and the photos of cell apoptosis were observed by transmission electron microscope. Telomeric Repeat Amplification Protocol (TRAP) combined with Enzyme Linked Immunosorbent Assay (ELISA) was used to detect the inhibitory rate of telomerase activity.Results:The obvious inhibitory effect of FC on human hepatocarcinoma cell lines (BEL 7402) was time and concentration dependent. The effect was significant when compared with the control group ( P <0.05). In addition,FC could induce cell apoptosis of hepatoma cell lines (BEL 7402) with a good dose dependent correlation. A marked arrestment at G 0/G 1 phase and a sub G 1 cell peak were observed. Meanwhile,the telomerase activity was down regulated significantly contrast to the control group ( P <0.05),the inhibitory rate has a positive correlation ( r=0.964,P <0.05) with the concentration of FC. Hepatocarcinoma cells cultured with different concentrations presented apoptosis features under electron microscope: intact cell membrane,chromatin condensation,nucleic fragmentation and apoptotic body formation. Conclusions:Our study demonstrated that FC could effectively inhibit the tumor cell proliferation,induce the cell apoptosis of the human hepatocarcinoma cell line (BEL 7402),and down regulate the telomerase activity. These effects may be responsible for its anti cancer mechanism.