携带甲胎蛋白基因小干扰RNA慢病毒载体的构建及其对靶基因的沉默效率

Construction of lentiviral vectors carrying small interference RNA of alpha-fetoprotein gene and its efficiency of target gene silencing

目的构建携带甲胎蛋白（AFP）基因小干扰RNA（siRNA）的慢病毒载体并转染肝癌细胞，评价其对AFP基因的沉默效率。方法设计和构建针对AFP基因的阳性siRNA及不针对任何已知基因的阴性siRNA，将其分别插入携带绿色荧光蛋白（GFP）基因的重组慢病毒载体，然后转染到表达AFP基因的人肝癌细胞HepG2并筛选阳性表达细胞株，分别为慢病毒转染阳性siRNA组和慢病毒转染阴性siRNA组。同时设立脂质体转染阳性siRNA组、脂质体转染阴性siRNA组以及加AFPsiRNA而未用转染试剂的siRNA组、空白对照组。荧光显微镜观察评估转染效率，荧光定量PCR和免疫印迹法检测各组HepG2细胞APFmRNA及蛋白的相对表达量，比较不同转染方法对AFP基因表达的抑制率。结果慢病毒能够有效地转染siRNA到HepG2细胞内。荧光定量PCR显示慢病毒转染siRNA对AFPmRNA表达的抑制率显著高于脂质体转染siRNA（92．1％比74．3％，P〈0．05）。免疫印迹亦显示慢病毒转染siRNA对AFP蛋白表达的抑制率显著高于脂质体转染siRNA（88．2％比63．7％，P〈0．05）。结论慢病毒转染AFPsiRNA能够更有效地抑制HepG2细胞内AFP基因表达。

Objective To construct lentiviral vectors carrying small interference RNA （siRNA） of alpha-fetoprotein（AFP） followed by transfection into hepatocellular carcinoma cells and to assess the efficiency of AFP gene silencing. Methods The AFP-specific positive siRNA and non-specific negative siRNA were designed and constructed prior to insertion to the recombinant lentiviral vectors carrying green fluorescent protein （GFP） gene. This was followed by transfection into human hepatocellular carcinoma cells HepG2 and subsequent screening of cell strains with GFP expression, and were assigned into lentivirus- transfected positive siRNA group and lentivirus-transfected negative siRNA group respectively. Furthermore, liposome-transfected positive siRNA group, liposome-transfected negative siRNA group, siRNA group which treated with AFP siRNA but without transfection reagent, and blank control group were established simultaneously. The efficiency of transfection was assessed under fluorescent microscope, and the relative expression of AFP mRNA and protein in HepG2 cells was determined via fluorescent quantitative polymerase chain reaction and immunoblotting assay. In addition, the inhibition rate of AFP gene expression was compared among all groups. Results Lentiviral vectors were associated with effective transfection of siRNA into HepG2 cells. The siRNA transfected with lentivirus yielded considerably higher inhibition rate of AFP mRNA than that with liposomes, as evidenced by fluorescent quantitative polymerase chain reaction （92.1% vs 74.3%, P〈0.05）. Immunoblotting assay showed that higher inhibition rate of AFP protein was in favor of siRNA transfected with lentivirus, but not with liposomes （88.2% vs 63.7%, P〈0.05）. Conclusion AFP siRNA transfected with lentivirus is associated with more effective inhibition of AFP expression in HepG2 cells.