共干扰Hpa、EP-CAM对人胆囊癌细胞株裸鼠模型的实验研究

Research on the effect of combination interference of Hpa and EP-CAM on human gallbladder cancer in nude mouse model

目的:研究miRNA转染乙酰肝素酶(heparanase,Hpa)和上皮细胞黏附分子(epithelial cell adhesion molecule,EP-CAM)对胆囊癌裸鼠移植瘤的体内抑瘤作用。方法:通过脂质体转染法将含Hpa、EP-CAM的真核表达质粒分别及共同转染至人胆囊癌细胞株GBC-SD,MTT法检测体外细胞增殖情况,筛选出干扰稳定表达细胞株后,采用裸鼠腋外侧皮下注射方法进行裸鼠体内成瘤实验,连续40 d观察裸鼠体表胆囊癌移植瘤生长情况。结果:单独及共同干扰组细胞生长受到明显抑制,而共同干扰组作用尤为突出。裸鼠体内实验提示Hpa miRNA组、EP-CAM miRNA组、共干扰组抑瘤率分别为69.06%、64.94%、85.67%。Hpa、EP-CAM基因蛋白在干扰组表达均明显低于正常胆囊癌细胞接种组(P<0.01)。结论:采用RNAi法沉默Hpa和EP-CAM在胆囊癌细胞中的表达可有效抑制其在体内外的生长,并且以共同干扰效果尤为突出。

Objective：To study the role of miRNA transfection of heparanase（Hpa） and epithelial cell adhesion molecule（EP-CAM） in inhibiting the growth or gallbladder cancer cells in nude mouse model.Methods：The Hpa and EP-CAM eukaryotic expression plasmids were single of respectively and co-transfected into the human gallbladder cancer cell GBC-SD by liposome transfection method.The cell proliferation was detected by MTT method.After separation of out the stable interference cells,the experiments in nude mice were completed by subcutaneously injecting gallbladder cells into the nude mice′s lateral axillary,and the growth of the gallbladder cancer tumor in nude mice was observed in the following forty days.Results：MTT experiments showed that the cell growth was inhibited in the single transfected group and the co-transfected group,particularly in the co-transfected group.Nude mice models experiments showed suggested that the inhibition rate was 69.06%,64.94% and 85.67% in Hpa miRNA group,EP-CAM miRNA group and co-transfected group respectively.Hpa and EP-CAM′s protein expressions in the interference groups were significantly lower than those in normal gallbladder cancer cells group.Conclusions：The method of using RNAi with Hpa and EP-CAM can inhibit the cell growth both in human gallbladder cancer cells and nude mouse model,and the effect is particularly prominent in the co-transfected group（P〈0.01）.