细胞内表达的小干扰RNA靶向丙肝病毒5′保守区的研究

将丙型肝炎病毒(HCV)基因组的5′非编码区(5′UTR)插入到报告基因绿色荧光蛋白(eGFP)和荧光素酶(luciferase)的上游,并构建基于Ⅲ型启动子的表达载体,这种载体能产生针对HCV 5′UTR的小干扰RNA。然后将含有HCV 5′UTR的eGFP/luciferase和能产生小干扰RNA的质粒共转染入Hela细胞,通过测定细胞发出的荧光和化学发光强弱来观测抑制效果。实验结果表明,与HCV 5′UTR特异性小干扰RNA表达质粒共转染的细胞无论从定性还是从定量上所测得的荧光和化学发光强度都明显低于阴性对照,且细胞密度经核染色与对照组无明显区别。这揭示了小干扰RNA确实能引起HCV特异基因如5′UTR的沉默,且转染进去的小干扰RNA表达质粒对细胞没有毒害作用。这一工作是通过载体直接在细胞内表达小干扰RNA(siRNA)而不是化学合成的,可以使小干扰RNA在细胞内得到稳定表达,因此本研究设计的siRNA表达载体不仅可以有效沉默HCV 5′UTR,而且该系统可以灵敏地筛选更有效的针对HCV的siRNA,因而这一结果为研究利用RNA干扰进行基因治疗HCV感染做了初步探索。

Targeting the 5' untranslated Region of Hepatitis C virus by Intracellularly Expressed Small Interfering RNA

In this study, we inserted the 5' untranslated region (UTR) of Hepatitis C virus (HCV) genome into the upstream of the reporter genes of enhanced green fluorescent protein (eGFP) and luciferase, and we also constructed the expression vector that can express the short interfering RNAs (siRNA) against the HCV 5' -UTR. The 5' -UTR-eGFP/luciferase and the siRNA-producing plasmid were cotransfected into Hela cells, and the inhibition effect was detected by the intensity of the fluorescence and luminescence. The vesults showed that the light from the cotransfected cells was obviously weaker than the negative control both in quality and in quantities, while density of the cotransfected cells had no difference with the control plasmid as detected by nucleus staining.This work demonstrated that certain siRNA can target the 5' UTR of HCV, while no toxic effect had been observed in the cells. This work is the basis for future research in which RNAi activity is supposed to be utilized in the gene therapy with the the HCV infection. The siRNA is expressed intracellularly by vectors instead of chemical synthesis, and a new method can be used as a model to quickly and safely screen effective siRNAs targeting HCV.