siRNA沉默AFP基因对肝癌细胞系 EGHC-9901 增殖的影响

 目的 建立稳定表达AFP-siRNA质粒的肝癌细胞系并探讨对其增殖的影响。方法 构建AFP-siRNA，用脂质体法转染肝癌细胞系，G418筛选4～5周，Western blot 及RT-PCR检测靶基因表达，分组：实验组，转染AFP-siRNA组；阳性对照组，转染空载体组；空白对照组，未处理组。MTT绘制生长曲线，平板克隆实验观察集落形成，流式细胞仪分析细胞周期。结果 成功建立稳定表达AFP-siRNA的肝癌细胞系，实验组表达AFP近乎完全抑制；实验组增殖能力显著低于空载体组；实验组＞75μm集落形成数19±2，低于空载体组62±6；实验组G1期细胞数较空载体组提高20％左右。 结论 成功建立稳定表达AFP-siRNA的肝癌细胞系，抑制AFP表达可使肝癌细胞发生G1期阻滞，明显抑制其增殖及集落形成能力。

Effects of AFP silencing on proliferation of hepatocellular carcinoma cell line EGHC-9901

Objective to observe effects of AFP gene silencing by siRNA on proliferation of hepatocellular carcinoma cell line EGHC-9901. Methods siRNA expressing plasmid aimed at AFP gene was firstly established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; after G418 positive clone selection for 4-5 weeks, AFP expression both of mRNA and protein were detected by semi-quantitative RT-PCR and western blot; cells then were divided into three groups: experimental group, AFP-siRNA transfected; vector control group, empty vector transfected; blank group, untreated. MTT and flat plate clone formation assay were applied to evaluated cell proliferation, and flow cytometry was employed to observe cell cycle. Results siRNA expressing plasmid aimed at AFP gene was successfully transfected into hepatocellular carcinoma cell line; AFP expression was almost completely inhibited. The experimental group proliferated at a significantly lower speed than the positive control group; The experimental group exhibited less clones with diameters more than 75μm than the positive control group, p<0.05; flow cytometry manifested approximately 20% more cells of the experimental group within G1 phase than those of the positive group, indicating that inhibition of AFP expression may cause G1 phase arrest. Conclusion siRNA expressing plasmid aimed at AFP gene was successfully stably transfected into hepatocellular carcinoma cell line EGHC-9901; AFP gene silenced by siRNA induces growth and colony formation inhibition of hepatocellular carcinoma cell line EGHC-9901.