人IL-1β重组表达载体在肝癌细胞的表达及对其NK细胞杀伤敏感性的影响  

Expression of recombinant IL-1β in H7402 cells down-regulated NK cell mediated cytotolysis

作　　者：梁淑娟[1] 潘继红[1] 牟东珍[1] 肖伟玲[1] 鞠吉雨[1] 

机构地区：[1]山东省潍坊医学院分子免疫学重点实验室,潍坊261042

出　　处：《中国免疫学杂志》2007年第8期688-692,共5页Chinese Journal of Immunology

基　　金：山东省中青年科学家科研奖励基金(2004BSB14074);教育部科学研究项目重点项目(205090);山东省卫生系统重点科技人才基金资助

摘　　要：目的:构建全长人IL-1β真核表达载体,转染H7402肝癌细胞,分析对其NK细胞杀伤敏感性的影响。方法:RT-PCR法扩增人IL-1β基因全长编码序列,经T-A克隆,构建pIRES2-EGFP-IL-1β重组表达载体,采用阳离子聚合物jetPEI的方法转染H7402肝癌细胞,G418筛选获得稳定表达的细胞克隆,RT-PCR分析IL-1β的表达水平,MTT方法分析转染前后NK细胞对肝癌细胞杀伤活性的变化。结果:从LPS处理的人外周PBMCs总RNA中扩增出IL-1β(大小约829 bp),先构建pMD18-IL-1β克隆载体,DNA序列鉴定正确后,利用Pfu DNA聚合酶将IL-1β基因亚克隆到pIRES2-EGFP中,构建真核表达载体pIRES2-EGFP-IL-1β,经PCR、限制性酶谱分析(BamHⅠ和EcoRⅠ)和DNA序列测定正确后,将其转染H7402肝癌细胞,G418筛选获得稳定表达IL-1β的细胞,与转染空载体的细胞相比,该细胞对NK-92细胞杀伤的敏感性显著降低,效靶比10∶1时下降了约30%。结论:促炎性细胞因子IL-1β能够显著增强肝癌细胞对NK细胞杀伤的抵抗性,可能是导致肝癌细胞发生天然免疫逃逸的重要机制。To analyze expression of human IL-1βin H7402 hepatoma cells on their sensitivity to NK cell mediated cytotolysis. Methods：Total RNA was isolated from LPS stimulated PBMCs from healthy donor, full length human IL-1β gene was obtained by RT-PCR, the product was cloned first into pMD18-T vector and then sub-cloned into eukaryotic expression vector pIRES2-EGFP,Recombinant vector pIRES2-EGFP-IL-1β was verified by PCR, restriction enzyme digestion with both BamH I and EcoR I , as well as DNA sequencing. Purified pIRES2-EGFP-IL-1β plasmid was stably transfected into H7402 hepatoma cells the cationic complex of jetPEI. The expression level of IL-1β was determined by RT-PCR. Cytotoxicity of NK-92 cells against H7402 cells before and after transfection was assessed by MTY assay. Results. IL-1β gene, a 829 bp of the expressing product was prepared from total RNA isolated from LPS treated PBMCs. The product was cloned into pMD18-T vector to construct pMD18-IL-1βrecombinant vector that was proved by PCR and DNA sequencing. The PCR products amplified from pMD18-IL-1β by Pfu DNA polymerase was then sub-cloned into eukaryotic expression vector pIRES2-EGFP to create recombinant IL-1β expression plasmid pIRES2-EGFP-IL-1β. After verification by PCR, restriction enzyme digestion and DNA equencing, purified pIRES2-EGFP-IL-1β was transfected into H7402 hepatoma cells by jetPEI, then selected in G418（final concentration 1 000 μ/ml） for at least 5 weeks to obtain novel cell lines that could stably express recombinant IL-1β These cells expressed high level of IL-1β and were more resistant to NK-92 mediated cytotoxicity than the cells transfected with pIRES2-EGFP. The cytotoxic activity to H7402 cells decreased about 30% at effector to target ratio of 10： 1. Conclusion： Expression of proinflammatory cytokine IL-1βby hepatoma cells significantly down-regulated their sensitivity to NK cell mediated cytolysis, hence provided an important mechanism for their escape from innate immunity by NK cells.

关 键 词：IL-1Β 肝癌细胞 NK细胞 天然免疫逃逸 促炎性细胞因子 

分 类 号：R392.1[医药卫生—免疫学]