摘要：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(1uciferase)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HCV5 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⋯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。 Abstract：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．an d we also constructed the expression vector that Can express the short interfering RNAs (siRNA) against the HCV 5 ．UTR．The 5 ．UTR．eGFP／luciferase an d the siRNA-prod ucing plasmid were cotran sfected into Hela cells，an d the inhibition efect was detected by the intensity of the fluorescence an d luminescence．Th e vesults showed that the light from the cotransfected cells was obviously weaker than the negative control both in quality an d in quan tities，while density of the cotran sfected cells had no diference with the control plasmid as detected by nucleus staining．TlliS work demonstratedthat certain siRNA Can targetthe 5 UTR ofHCV whilenotoxic efecthadbeenobserved in the cells．TlliS 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，an d a new method Can be used as a model to quickly an d safely screen effcctive siI AstargetingHCV

Abstract：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．an d we also constructed the expression vector that Can express the short interfering RNAs (siRNA) against the HCV 5 ．UTR．The 5 ．UTR．eGFP／luciferase an d the siRNA-prod ucing plasmid were cotran sfected into Hela cells，an d the inhibition efect was detected by the intensity of the fluorescence an d luminescence．Th e vesults showed that the light from the cotransfected cells was obviously weaker than the negative control both in quality an d in quan tities，while density of the cotran sfected cells had no diference with the control plasmid as detected by nucleus staining．TlliS work demonstratedthat certain siRNA Can targetthe 5 UTR ofHCV whilenotoxic efecthadbeenobserved in the cells．TlliS 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，an d a new method Can be used as a model to quickly an d safely screen effcctive siI AstargetingHCV