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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: To construct an eukaryotic expression vector for the interleukin receptor 1 receptor IL1RA gene of interleukin receptor 1 and transfect it into human esophageal cancer cell lines to investigate the relationship between IL1RA and esophageal cancer. Methods: IL1RA was amplified from normal esophageal tissue by PCR and IL1RA was recombined into eukaryotic expression vector pcDNA3.1 to construct the eukaryotic expression vector pcDNA3.1IL1RA of IL1RA. Then the recombinants were introduced into human esophageal cancer cell line EC9706. The transfected cells were screened and identified by RTPCR and Southern hybridization to obtain positive clones. The effect of IL1RA on the growth of EC9706 cells was analyzed by cell counting and flow cytometry (FCM). RESULTS: RTPCR and Southern blot showed that IL1RA was highly expressed in transfected cell clones. Compared with EC9706 cells and pcDNA3.1 empty vector control cells, EC9706 cells transfected with pcDNA3.1IL1RA were due to IL1RA. Expression, cell proliferation rate has declined to varying degrees; FCM results show that IL1RA can not induce apoptosis of EC9706 cells, the effect on cell growth also showed inconsistency. Conclusion: It is suggested that IL1RA may only play a supporting role in the occurrence of esophageal cancer.