肝细胞LO2中细胞因子诱导的含CISH基因的表达及其炎性因子调控的研究  

A Study on the Gene Expression of Cytokine-inducible SH2-containing Protein(CISH)in Hepatocyte LO2 and the Regulation of Its Inflammatory Factors

作　　者：李腊梅[1] 杨雪梅[2] 伍洋平 陈洁[2] 胡章勇[2,1] 

机构地区：[1]成都医学院基础医学院,成都610083 [2]成都医学院第一附属医院,成都610500 [3]四川大学生物治疗国家重点实验室,成都610411

出　　处：《成都医学院学报》2017年第1期44-48,共5页Journal of Chengdu Medical College

基　　金：四川省科技厅资助项目(No:2016JY0207)

摘　　要：目的建立SH2结构域蛋白(cytokine inducible SH2-containing protein,CISH)基因上、下调的肝细胞模型,探讨CISH基因在正常肝细胞中的表达及其对炎性因子的调控。方法实验组分别为转染CISH siRNA组和CISH过表达质粒组,对照组分别为转染空质粒的阴性对照组和未转染任何物质的空白对照组;分别用CISH siRNA和CISH过表达质粒借助lipo 2000转染肝细胞LO2构建CISH基因表达的上、下调肝细胞模型;采用实时荧光定量PCR和蛋白质印迹法(Western Blot)分别检测肝细胞LO2中CISH基因在mRNA和蛋白水平的变化;利用ELISA法检测LO2肝细胞中CISH基因表达水平改变后对炎症因子TNF-α、IL-1β水平的影响。结果实时荧光定量PCR和Western Blot法检测表明,与未转染的LO2肝细胞比较,被转染CISH过表达质粒的细胞能够上调目的基因CISH的表达,被转染CISH siRNA的细胞可下调目的基因CISH的表达;ELISA法检测表明,与未转染的LO2肝细胞比较,被转染CISH过表达质粒的细胞分泌的炎症因子TNF-α和IL-1β水平表现出降低趋势,而被转染CISH siRNA的细胞分泌的炎症因子TNF-α和IL-1β水平则表现出升高趋势。结论成功构建了CISH高、低表达的LO2肝细胞系,CISH基因表达改变可影响LO2肝细胞系炎性因子的分泌。Objective To establish the hepatocyte models up-regulated and down-regulated by cytokine-inducible SH2-containing protein （CISH）gene and investigate the expression of CISH gene in the normal hepatocyte and the regulation of its inflammatory factors.Methods The experiment groups consisted of the groupinfected by CISH siRNA and that infected by CISH over-expression plasmid;while the control groups involved the negative group infected by empty plasmid and the uninfected blank group.The hepatocyte LO2 was transfected with CISH over-expression plasmid and CISH siRNA by Lipo 2000to establish the hepatocyte models up-regulated and down-regulated by CISH gene.Real-time Fluorescence Quantitative PCR and Western Blotting were used to detect the level changes of CISH mRNA and protein and Enzyme-linked immunosorbent assay （ELISA）was adopted to detect the effect of the CISH gene expression change on the levels of the inflammatory factors TNF-αand IL-1βin hepatocyte LO2.Results According to the results of Real-time Fluorescence Quantitative PCR and Western Blotting,the cells infected by CISH over-expression plasmid up-regulated the expression of CISH gene and those infected by CISH siRNA down-regulated the expression of CISH gene when compared with the uninfected hepatocyte LO2.The results of ELISA indicated that the levels of the inflammatory factors TNF-αand IL-1βsecreted by the cells infected by CISH over-expression plasmid decreased and those secreted by the cells infected by CISH siRNA increased when compared with the uninfected hepatocyte LO2.Conclusion Thehepatocyte LO2 cell lines up-regulated and down-regulated by CISH gene are established successfully,and the changes of the CISH gene expression can affect the secretion of inflammatory factors in hepatocyte LO2 cell lines.

关 键 词：慢性乙肝 肝细胞LO2 CISH基因 炎症因子 

分 类 号：R363.1[医药卫生—病理学]