蜂毒素对胃癌细胞系SGC-7901细胞增殖抑制作用及机制

Experimental study of the effects of mellitin on cell growth inhibition of gastric cancer SGC-7901 cells

目的研究蜂毒素影响人胃癌细胞SGC-7901增殖及凋亡的机制。方法使用不同浓度(1、2、4、8、16、32μg/ml)蜂毒素处理SGC-7901细胞不同时间(12、24、36、48、72h)后,采用MTT法测定肿瘤细胞生长抑制率;透射电镜观察细胞超微结构的改变;琼脂糖凝胶电泳观察凋亡细胞特征性DNA条带;流式细胞仪法检测细胞凋亡和细胞周期;免疫组化观察Bcl-2基因和Bax基因表达情况;RT-PCR检测Bcl-2、Bax、p53mRNA的表达。结果蜂毒素对胃癌SGC-7901细胞的生长抑制作用随着其作用时间的延长和浓度的增加而明显增强,作用48h后,其IC50为2.43μg/ml;透射电镜观察显示蜂毒素组细胞呈凋亡表现,核固缩、碎裂,染色质浓缩,边集;蜂毒素(4、8μg/ml)组培养48h后DNA琼脂糖凝胶电泳可见典型的梯状条带,而对照组细胞DNA未见梯状条带;流式细胞DNA直方图上出现典型的亚二倍体"凋亡峰",2、4、8μg/ml浓度蜂毒素作用人胃癌细胞后的细胞凋亡率分别为(5.69±0.74)%、(10.58±1.32)%、(29.57±1.58)%。Bax、p53基因mRNA和蛋白表达降低、Bcl-2mR-NA和蛋白表达升高,并随蜂毒素浓度增加而加强。结论蜂毒素能诱导人胃SGC-7901细胞凋亡和抑制增殖,且有一定的量效关系,其作用机制可能与调控Bcl-2、Bax、p53基因表达有关。

Objective To investigate the effects of mellitin on cell proliferation and apoptosis mechanism of gastric adenocarcinoma SGC-7901 cells.Methods SGC-7901 cells were treated with different concentrations（1,2,4,8,16 and 32 μg/ml）of mellitin for various periods（12,24,36,48 and 72 h）.The cell proliferation was examined by MTT assay.Ultramicro structure was observed through transmission electron microscope（TEM）.Gel electrophoresis was used to observe the typical DNA ladder.Apoptosis and cell cycle were determined by flow cytometry（FCM）.Expressions of Bcl-2,Bax proteins in gastric cancer cells were measured by immunohistochemistry.RT-PCR was used to detect the mRNA transcriptions of Bcl-2,Bax and p53.Results Melltin inhibited the growth of gastric adenocarcinoma SGC-7901 cells in time-and concentration-dependent manners with IC50 value of 2.43 μg/ml（treated with mellitin after 48 h）.The apoptotic morphology,such as karyopycnosis and apoptotic body,could be observed by TEM in mellitin groups.The 4,8μg/ml mellitin groups produced characteristic DNA ladder on DNA gel,but not in the control and a tipical subdiploid peak before phase G0/G1was detected by flow cytometry.The rates of apoptosis were correlated with the concentration of mellitin,after treated with 2,4,8μg/ml of mellitin for 48 h,the cell apoptotic rates were respectively 5.69%±0.74%,10.58%±1.32% and 29.57%±1.58%. The expression of Bax and p53 mRNA and Bax protein obviously increased respectively.The expression of Bcl-2 mRNA and Bcl-2 protein obviously decreased respectively.Conclusion Mellitin can induce apoptosis and inhibit proliferation of gastric adenocarcinoma SGC-7901 cells and display effect in a dose-dependent manner by regulating expression of Bcl-2,Bax,p53 genes.