诱导型一氧化氮合酶基因启动子在不同细胞系中的转录活性  

Transcription activation of Inducible Nitric Oxide Synthase gene in different cell lines

作　　者：郭风劲[1] 成军[2] 纪冬[2] 刘妍[2] 王琳[2] 张黎颖[2] 戴久增[2] 宋方洲[3] 

机构地区：[1]重庆医科大学生物学教研室,重庆市400016 [2]北京地坛医院传染病研究所,北京市100011 [3]重庆医科大学分子生物学教研室,重庆市400016

出　　处：《世界华人消化杂志》2005年第4期460-463,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30371288北京市自然科学基金资助项目;No.5042024国家人事部第35批博士后科学基金资助项目;No.2004035045~~

摘　　要：目的:探讨诱导型一氧化氮合酶(iNOS)基因启动子在不同细胞系中转录活性的差别. 方法:利用生物信息学技术确定iNOS基因的启动子区域(iNOSp),聚合酶链反应(PCR)扩增iNOSp,克隆至真核报告载体pCAT3-Basic中,构建pCAT3-iNOSp报告载体;以该质粒分别转染正常人肝细胞株LO2,人肝母细胞瘤细胞系HepG2,小鼠成纤维细胞NIH-3T3, 人贮脂细胞(Lipocyte Ito Cell)和肝癌细胞株SMMC- 7721,用ELISA法检测氯霉素乙酰转移酶(CAT)在不同细胞系中的表达活性. 结果:成功获得iNOS基因启动子的正确克隆,克隆的iNOS启动子有顺式激活下游基因的活性.pCAT3- iNOSp瞬时转染HepG2细胞的CAT表达活性最强,肝贮脂细胞Ito中CAT表达活性最弱. 结论:不同细胞中iNOS启动子的表达活性不同,其表达活性与细胞类型与细胞状态密切相关.AIM: To investigate the transcription activation of induc-ible nitric oxide synthase (iNOS) gene in different cell lines. METHODS: The coding sequence of iNOS promoter (iNOSp) was amplified by PCR from genomic DNA of human hepatoblastoma cell line HepG2, and cloned into pEGM-Teasy vector to yield Teasy- iNOSp. The iNOSp gene was cut from Teasy-iNOSp by Kpnl and Xhol, and then subcloned into pCAT3-Basic to produce a construct named pCAT3-iNOSp. pCAT3-iNOSp was transfected into HepG2cell line, SMMC-7721 human hepatocellular carcinoma cell line, NIH-3T3 mouse fibroblast cell line, Ito human lipocyte cell line, and L02 human normal liver cell line by FuGENE 6 transfection reagent. Cells transfected with pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of CAT, which reflects the transcription activation of the iNOS gene promoter, was detected by ELISA after 48 hours of transfection. RESULTS: The report vector pCAT3-iNOS was successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activity of CAT was highest in HepG2 cells transfected with pCAT3-iNOSp, and lowest in Ito cells. CONCLUSION: The iNOS gene promoter can transactivate its downstream genes. Its transcription activity varies in different cell lines.

关 键 词：INOS基因 诱导型一氧化氮合酶 细胞系 启动子 转录活性 贮脂细胞 转染 表达活性 报告载体 克隆 

分 类 号：R346[医药卫生—基础医学]