RNA干扰抑制HepG2细胞AFP基因表达实验方法建立

Establish the Method of RNA Interference Inhibiting AFP Gene expression in HepG2

目的:筛选较理想的阳离子脂质体META/pGenesil-AFP质粒转染HepG2的方法。方法:将针对两条靶序列AFP1和AFP2的shRNA的DNA模板双链,连接于质粒表达载体,构建质粒载体pGenesil-1-AFP1(含绿色荧光蛋白GEP编码序列)及pGenesil-2-AFP2,用阳离子脂质体META/质粒载体pGenesil-1-AFP1转染HepG2细胞。荧光显微镜观察并计算不同剂量配比的脂质体/质粒载体混合液转染效果及微粒子化学发光免疫分析法检测不同剂量配比转染后对AFP基因表达的影响。结果:剂量比为8μL∶2.5μg条件,能有效地转染HepG2细胞,并有效抑制AFP表达且无细胞毒性作用。结论:本研究成功建立了阳离子脂质体META/质粒载体pGenesil-1-AFP1转染HepG2细胞实验方法。

Object：To establish the new method of transfecting HepG2 cells with pC, enesil and Metafectence lipofection regent complex. Methods：pGenesil - 1 -AFP1 （AFP2） was constructed by inserting AFP1 - or AFP2 -targeted shRNA into plasmid expression vector, then transfected into HepG2 cells via lipofection. The transfection effect observed by fluorescent microscope and AFP gene expression was assayed by microbeads - based chemiluminescence immunoassay. Result：The optimal ratio of pC, enesil and Metafectence lipofection regent complex was 8 μL ：2.5 μg. Conclusion：The method of transfecting HepG2 cells via lipofe.ction was successfully established.