反义RNA体外抑制丙型肝炎病毒基因表达的研究  

Inhibition of hepatitis C virus gene expression by antisense nucleotide in vitro

作　　者：李勇年[1] 于敏[2] 吴炜强[1] 高建兴[1] 王洪[1] 纪绍平[3] 王勤环[2] 斯崇文[2] 

机构地区：[1]解放军第三二三医院感染科,西安710054 [2]北京大学第一医院感染科 [3]第四军医大学生化教研室

出　　处：《中华实验和临床病毒学杂志》2004年第4期341-343,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的　研究丙型肝炎病毒 (HCV)基因调控方式及反义RNA对HCV基因表达的体外抑制作用。方法　采用业已建立的HCV基因调控细胞模型 ,以重组质粒共转染策略 ,将反义RNA重组质粒与HCV 5′非翻译区 ( 5′UTR )调控的报道基因———虫荧光素酶 (luc)重组质粒共同转染人肝癌细胞系 (HepG2 ) ,并以不表达反义RNA的原核重组质粒和不含有HCV 5′UTR的luc重组质粒做为对照。转染细胞经短期培养后制备细胞提取液 ,荧光检测法检测luc基因的表达。结果　针对HCV 5′UTR的反义RNA可以在体外有效抑制由HCV 5′UTR调控的luc基因的表达 ,并具有剂量依赖效应。对照重组质粒转染实验证明 ,上述抑制作用呈序列特异性。结论　HCV 5′UTR具有调控下游目的基因表达的重要功能 ,反义RNA可以在体外有效抑制由HCVObjective To study the mechanism of hepatitis C virus (HCV) gene regulation and the inhibitory effect of antisense RNA on HCV gene expression in vitro . Methods The hepatoblastoma cell line (HepG 2) was co-transfected by recombinant plasmid of antisense RNA complementary to HCV 5′ untranslated region (5′ UTR)and HCV 5′ UTR Directed luciferase (luc) gene expression recombinant plasmid. Meanwhile a reversed HCV 5′UTR recombinat plasnid which can not transcribe as antisense RNA in the cell and a recombinant plasmid in which the luc was regulated by simian virus 40 (sv40) 5′UTR were used as controls respectively. The level of luc gene expression was determined by an enzymatic assay. Results The antisense RNA which directed to HCV 5′UTR could obviously knock down the level of luc gene expression and the close-dependent inhibition of antisense RNA was observed at the same time. However the above inhibition was not shown in the cells co-transfected by reversed HCV 5′UTR recombinant plasmid and HCV 5′UTR directed luc gene expression recombinant plasmid. No reduction was obserred in luc gene expression level in the cell co-transfected by both antisense RNA recombinant plasmid and SV 40 5′UTR directed luc gene expression recombinant plasmid. Conclusion HCV 5′UTR plays an important role in regulation of viral gene expression. The antisense RNA complementary to HCV 5′UTR could effectively inhibit the gene expression regulated by HCV 5′UTR in vitro .

关 键 词：HCV 反义RNA 基因表达 丙型肝炎病毒 重组质粒 体外抑制 C基因 基因调控 目的基因 虫荧光素酶 

分 类 号：R346[医药卫生—基础医学]