表达谱基因芯片技术研究甘草甜素上调白介素-18基因的表达  

Up-regulating effects of glycyrrhizin on interleukin-18 gene expression by cDNA microarray

作　　者：王建军[1] 李宝伟[2] 成军[1] 刘妍[1] 徐志强[1] 杨倩[1] 纪冬[1] 党晓燕[1] 王春花[1] 

机构地区：[1]中国人民解放军第302医院传染病研究所基因治疗研究中心,全军病毒性肝炎防治研究重点实验室,北京市100039 [2]中国人民解放军总医院麻醉科,北京市100853

出　　处：《世界华人消化杂志》2004年第4期855-858,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金攻关项目;No.C03011402;No.C30070689军队"九;五"科技攻关项目;No.98D063军队回国留学人员启动基金项目;No.98H038军队"十;五"科技攻关青年基金项目;No.01Q138军队"十;五"科技攻关面上项目;No.01MB135~~

摘　　要：目的:了解甘草甜素调节白介素-18(IL-18)基因启动子的活性,为甘草甜素的作用机制提供理论依据. 方法:应用基因表达谱芯片技术对于甘草甜素刺激HepG2 细胞之后的基因表达谱进行研究.聚合酶链反应(PCR)扩增IL-18基因启动子,命名为IL-18P.以T-A克隆法,将IL- 18P基因片段连入载体pGEM-T.将获得的质粒pT-IL- 18P,与报告质粒pCAT3-basic分别用KpnⅠ和BglⅡ双酶切后构建IL-18启动子报告基因表达载体pCAT3-IL-18P, 以重组表达质粒pCAT3-IL-18P瞬时转染HepG2细胞,以转染pCAT3 basic的HepG2细胞为阴性对照,甘草甜素刺激后24 h后收获细胞.用酶联免疫黏附法(ELISA)检测细胞中氯霉素乙酰转移酶(CAT)的表达活性. 结果:表达谱基因芯片研究结果表明,甘草甜素可上调IL- 18基因表达水平达2.815倍.构建的报告载体pCAT3-IL- 18P经过序列分析和酶切鉴定正确.pCAT3-IL-18P和甘草甜素瞬时转染的HepG2细胞的CAT表达活性是CAT3空载体的7.7倍,pCAT3-IL-18P的1.5倍. 结论:甘草甜素可以上调IL-18启动子的活性,进而上调IL-18基因的表达.为深入了解甘草甜素的免疫调节作用及其在病毒的清除过程中的作用机制提供新的理论依据.AIM: To investigate the relationship of glycyrrhizin and interleukin 18 (IL-18) gene expression and to explore the molecular biological mechanisms of glycyrrhizin in antivirus functions. METHODS: cDNA microarray was used for the study of up-regulated gene by glycyrrhizin. Polymerase chain reaction (PCR) technique was employed to amplify the sequence of IL-18 promoter by using HepG2 cell genomic DNA as the template, named IL-18P, and the PCR product was cloned into pGEM-T vector. The IL-18P gene was cut from T-IL-18 P by KpnⅠ and Bgl Ⅱ, and then cloned into pCAT3 basic, named pCAT3- IL-18P. pCAT3-IL-18P was transfected into the HepG2 cell line and cotransfected HepG2 cells with glycyrrhizin by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-basic was used as negative control. The activity of chloramphenicol acetyltransferase (CAT) in HepG2 cells transfected was detected by an enzyme-linked immunoassay (ELISA) kit after 48 h, which reflected the transactivating function of glycyrrhizin to IL-18 gene promoter. RESULTS: The expressive vector pCAT3-IL-18P was constructed and confirmed by restriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells transfected with pCAT3-IL-18P and stimulated with HepG2 was 7.7 times as higher as that of pCAT3-basic, and 1.5 times as higher as that of pCAT3-IL-18P. CONCLUSION: HepG2 can up-regulate IL-18 gene promoter. These results provide a new evidence to explain the molecular biological mechanisms of HepG2 in immuno-regulation and antivirus.

关 键 词：表达谱 基因芯片技术 甘草甜素 白介素-18 基因表达 酶联免疫黏附法 ELISA 

分 类 号：R285[医药卫生—中药学]