Title: Expression of recombinant IL-1β in H7402 cells down-regulated NK cell mediated cytotolysis
Abstract: Objective:To analyze expression of human IL-1β in 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 MTT 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-1β recombinant 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 sequencing,purified pIRES2-EGFP-IL-1β was transfected into H7402 hepatoma cells by jetPEI,then selected in G418(final concentration 1 000 μg/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-1β by 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.
Publication Year: 2007
Publication Date: 2007-01-01
Language: en
Type: article
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