HCVIRES调控外分泌性碱性磷酸酶基因在肝细胞中的表达  

Expression of secreted alkaline phosphatase inhepatocytes controlled by HCV IRES

作　　者：刘水平[1] 谭德明[1] 侯周华[1] 刘双虎[1] 文质[2] 

机构地区：[1]中南大学湘雅医院传染病研究所,长沙410008 [2]中南大学湘雅医学院微生物学教研室,长沙410078

出　　处：《湖南医科大学学报》2003年第4期335-337,共3页Bulletin of Hunan Medical University

基　　金：省医药卫生科技项目 (2 0 0 0 0 0 1) ;湖南省科技攻关项目 (0 2ssy30 83)

摘　　要：目的 :构建HCVIRES调控外分泌性碱性磷酸酶 (SEAP)基因表达的细胞模型。方法 :用PCR技术扩增HCV 5′NCR片段 ,定向克隆至表达质粒 pSEAP2 Control的SEAP基因上游 ,构建HCV 5′NCR调控SEAP表达的重组质粒 pdNCRSEAP。用脂质体基因转染技术 ,将pdNCRSEAP转染至肝细胞株QSG770 1。用化学发光法检测SEAP的表达 ,并观察不同浓度反义寡聚核苷酸 (ASODN)对SEAP表达的影响。结果 :重组质粒 pdNCRSEAP表达的发光强度为pSEAP2 Control的 76 %,5 μmol和 10 μmolASODN对pdNCRSEAP发光强度的抑制率分别为2 9.2 %和 44 .6 %,而对 pSEAP2 Control的SEAP表达无显著抑制作用 (P >0 .0 5 )结论 :重组质粒 pdNCRSEAP的SEAP表达受HCV 5′NCR调控 ,为以HCV 5′NCR为靶位点的药物评价建立了检测方便的细胞模型。Objective To establish a cell model of secreted alkaline phosphatase (SEAP) controlled by HCV internal ribosome entry site ( IRES ). Methods The fragment of HCV 5′ noncoding region (5′ NCR ) was amplified by polymerase chain reaction ( PCR ), and was immediately cloned the upstream of the SEAP gene of pSEAP2-Control, an SEAP eukaryotic expression plasmid. With the liposome transfection technique, the resulting recombinant plasmid pdNCRSEAP was transfected into hepatocytes QSG7710, and the SEAP activity of cell culture media was monitored quantitatively by the chemiluminescent method. The regulatory effect of the HCV 5′ NCR on the SEAP expression was measured by the treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) at 5 μmol and 10 μmol, respectively. Results The light emission intensity of pdNCRSEAP expression was 76% that of pSEAP2-Control. The inhibition rates of pNCRSEAP luminescence intensity affected by ASODN of 5 μmol and 10 μmol were 29.2% and 44.6%, respectively, while ASODN had no significant effect on the pSEAP2-Control expression. Conclusion The SEAP expression of pdNCRSEAP is controlled by HCV 5′NCR. The cell model of drug evaluation targeted at HCV 5′NCR is successfully established and can be analyzed conveniently.

关 键 词：HCV IRES 调控 外分泌性碱性磷酸酶基因 肝细胞 表达 

分 类 号：R346[医药卫生—基础医学]