IL-1受体拮抗物基因克隆对人食管癌细胞株生长的影响

Cloning of IL-1RA Gene and Its Effect on Human Esophageal Cancer Cells

目的:构建白细胞介素1受体拮抗物interleukin1receptorantagonistIL1RA基因的真核表达载体,并转染人食管癌细胞株,以探讨IL1RA与食管癌发生的关系。方法:利用PCR方法从正常食管组织cDNA中扩增IL1RA,将IL1RA重组到真核表达载体pcDNA3.1中,构建IL1RA的真核表达载体pcDNA3.1IL1RA。然后将重组子导入人食管癌细胞株EC9706中,用RTPCR及Southern杂交的方法,筛选鉴定转染细胞,获得阳性克隆。并采用细胞计数法和流式细胞术(FCM)分析IL1RA对EC9706细胞生长的影响。结果:RTPCR及Southern杂交结果显示转染后的细胞克隆均呈现IL1RA高表达,与对照组EC9706细胞和带有pcDNA3.1空载体的细胞相比,转染有pcDNA3.1IL1RA的EC9706细胞由于IL1RA的表达,细胞增殖速度有不同程度的下降;FCM结果显示IL1RA不能诱导EC9706细胞的凋亡,对细胞生长的影响亦呈现不一致性。结论:提示IL1RA对食管癌的发生可能只起辅助作用。

Objective:This current study was designed to construct the mammalian expression pl asmid of interleukin1-receptor antagonist(IL-1RA)and investigate the effect of IL-1RA on human esophageal cancer cell.Methods :A full length human IL-1RA DNA was amplifie d from cDNA of the normal esophageal t issue by polymerase chain reaction(PCR)method,and was inserted into a mamma lian expression vector pcDNA3.1to m ake an IL-1RA expression plasmid pcDNA3.1-IL-1RA.Then the recombin ant was transfected into human esophageal cancer cells EC9706.The posit ive clones were selected by RT-PCR method and Southern blot.Counting method and FCM analysis were performed to measure the effect of IL-1RA on EC9706cells.Results:This study showed that every clone of EC9706with pcDNA3.1-IL-1RA,which was identif ied by both RT-PCR and Southern blot,has a high level expression of IL-1RA after transfection.Comparing with contr ol EC9706cells and EC9706transfected with pcDNA3.1vector alone,the growth rate of different clones of EC9706with pcDNA3.1-IL-1RA was suppressed variously for the expression of IL-1RA.The r esult of FCM analysis showed that the changes in cell phases of the cells with pcDNA3.1-IL-1RA were different as well as no significant apoptosis can be observ ed in the transfected cells.Conclusions :The results imply that IL-1RA may be a subsidiary gene in the process of eso phageal cancer,may have cooperative effect with other genes.