小鼠IL-1β在H22细胞的表达及对NK细胞杀伤活性的影响  

Expression of recombinant mouse IL-1β significantly down-regulates NK cell mediated cytotolysis against H22 hepatoma cells

作　　者：肖伟玲[1] 梁淑娟[1] 牟东珍[1] 王雪净[1] 吴慧娜[1] 

机构地区：[1]潍坊医学院分子免疫学重点实验室,山东潍坊261042

出　　处：《细胞与分子免疫学杂志》2007年第12期1130-1132,1135,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30772497);教育部科学技术研究重点项目(205090);山东省中青年科学家科研奖励基金资助项目(2004BSB14074);山东省卫生高层次人才1020工程专项基金资助项目(2006)

摘　　要：目的:重组表达小鼠IL-1β全长基因,转染H22肝癌细胞,分析对NK细胞杀伤活性的影响。方法:构建小鼠IL-1β重组表达载体pIRES2-EGFP-mIL-1β,利用jetPEI转染H22肝癌细胞,RT-PCR和激光扫描共聚焦显微镜分析IL-1β重组载体的表达,MTT方法分析转染前后,野生型小鼠脾脏NK细胞对H22细胞杀伤活性的变化。结果:RT-PCR扩增出小鼠IL-1β,长度约843bp。纯化的PCR产物与pIRES2-EG-FP同时经XhoI和EcoRI双酶切,在T4连接酶作用下连接,转化大肠杆菌,提取质粒经PCR、限制性酶谱分析(XhoI+EcoRI)和DNA序列测定后,确认获得重组表达载体pIRES2-EGFP-mIL-1β。将该质粒转染至H22小鼠肝癌细胞中,RT-PCR和荧光观察证实,H22细胞能表达高水平IL-1β重组表达载体,与转染空载体的对照组细胞相比,IL-1β转基因后H22细胞对NK92细胞杀伤抵抗性明显增强,同时野生型小鼠脾脏NK细胞对pIRES2-EGFP-mIL-1β转基因细胞的杀伤活性明显下降,效靶比40:1时下降了约10%。结论:IL-1β能够明显增强H22肝癌细胞对NK细胞杀伤的抵抗性,可能是肝癌细胞借以逃逸天然免疫应答的重要机制。AIM： To investigate the effect of the expression of recombinant IL-1β in H22 hepatoma cells on its response to NK cell mediated cytotoxicity. METHODS： BALB/c mouse was stimulated by 6% of starch. Total RNA was prepared from peripheral blood monocytes （PBMCs）. IL-1β gene （843 bp） was obtained by RT-PCR. The purified PCR product digested by Xho I and EcoR I was cloned into plRES2-EGFP to construct the recombinant plRES2-EGFP-mIL-1β expression vector which was verified by PCR, restriction enzyme assay （Xho I and EcoR I） and DNA sequencing. Then the purified plRES2-EGFP-mIL-1β plasmid was transfected into H22 hepatoma cells by jetPEI. The expression level of recombinant IL-1β was detected by RTPCR and confocal microscopy. The cytotoxicity of wild-type spleenic NK cells against H22 cells was assessed by MTT assay. RESULTS： After the total RNA isolated from the starch stimulated BALB/c mouse PBMC, 843 bp IL-1β gene in length was prepared by RT-PCR. The purified PCR product digested by EcoR I and Xho I was ligated by plRES2- EGFP to create plRES2-EGFP-mIL-1β expression plasmid which was verified by PCR, restriction enzyme assay and DNA sequencing. Then plRES2-EGFP-mIL-1β was transfected into H22 hepatoma cells by jetPEI. RT-PCR and confocal microscopy assay showed these cells expressed high level of recombinant IL-1β expression vector. In a 4-hour based MTT assay, IL-1β in H22 cells was more resistant to NK92 cell mediated cytotoxicity compared with the cells transfected with plRES2-EGFP. Meanwhile, the cytolytic capacity of the spleenic NK cells separated from wild-type mouse decreased about 10% when the ratio of effector to target was 40： 1. CONCLUSION： The expression of proinflammatory cytokine IL-1β can significantly down-regulate the cytolytic activity of NK cells against H22 hepatoma cells. It plays a crucial role in the immune escape of hepatoma from NK cell mediated innate immunity.

关 键 词：IL-1Β NK细胞 天然免疫 肝癌 

分 类 号：R392.12[医药卫生—免疫学]