TNFα基因在小鼠成纤维细胞L929中的调控研究

The Regulation Effect of TNFα Gene in Murine Fibroblast L929

质粒pEGFP 1是一种不含启动子序列的哺乳动物细胞表达载体 .将系列缺失的TNFα基因启动子、5′UTR(untranslatedregion)和 3′UTR插入 pEGFP 1中 ,构建了一组重组质粒 .转染L92 9细胞后 ,发现当报告基因EGFP的上游含有包括 5′UTR在内的TNFα基因启动子序列时 ,重组质粒在L92 9细胞中均能表达EGFP .但是 ,当EGFP基因 3′端同时含有TNFα基因的 3′UTR时 ,重组质粒转染L92 9细胞后 ,均不能表达EGFP .因此 ,在L92 9细胞中 ,TNFα基因的 3′UTR对基因表达具有抑制作用 .进一步研究发现 ,在L92 9细胞中 ,TNFα基因的 3′UTR对基因表达的抑制作用需要其 5′UTR的共同参与 ,而且 ,RT PCR实验表明 ,LPS在其他细胞中对TNFα表达起诱导作用的机制在L92

Plasmid pEGFP 1 is a mammalian cell expression vector whose reporter gene lacks its own promoter sequence. A series of constructs consisting of EGFP report gene flanked by the TNFα promoter,5′ untranslated region (UTR) and 3′UTR were constucted. After transient transfection of the constructs containing the TNFα promoter sequence including 5′UTR, EGFP gene is constitutively expressed in L929 cells, but EGFP gene become inactive by the insertion of the 3′UTR into the 3′end of EGFP report gene. The results suggested the inhibitory effect of the 3′UTR of TNFα gene on gene expression in L929 cells. The inhibitory effect of 3′UTR of TNFα gene on gene expression in L929 cells needs in synergy with its 5′UTR. Furthermore, the activation effect of lipopolysaccharide (LPS) on TNFα gene expression is absent in L929 cells detected by RT PCR analysis.