携人IL-12基因腺病毒的构建及其对Hep3B细胞增殖及TGF-β表达的影响  

Construction of adenovirus vector containing hIL-12 gene and its effects on the proliferation and TGF-β expression of Hep3B cells

作　　者：成凤[1] 匡文斌[1] 王秦[1] 秦晓林[1] 范晓卿[1] 董晋豫[1] 梁勤东[1] 李朴[1] 涂植光[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点试验室,重庆400016

出　　处：《中国免疫学杂志》2012年第5期402-406,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(81172016)资助

摘　　要：目的:构建携带人IL-12的重组腺病毒,观察其对Hep3B增殖及TGF-β表达的影响。方法:PCR扩增IL-12基因,构建穿梭质粒pAdTrack-IL-12;穿梭质粒经Pme I线性化后转化AdEasier Cell,E.coli BJ5183内同源重组;PacⅠ酶切重组腺病毒质粒,转染AD293,包装病毒;病毒pAd-IL-12感染肝癌细胞Hep3B,以RT-PCR、Western blot检测白介素12的表达;MTT法检测肝癌细胞的增殖情况;RT-PCR检测转化生长因子-β(TGF-β)的表达。结果:成功构建携IL-12基因的重组质粒,并成功包装可高效感染Hep3B的腺病毒pAd-IL-12;RT-PCR以及Western blot结果表明,病毒pAd-IL-12感染Hep3B后可高表达白介素12,并且与对照组相比Hep3B增殖能力显著降低(P<0.05),TGF-β表达量也降低(P<0.05)。结论:可高表达IL-12的腺病毒包装成功,感染肝癌细胞Hep3B后抑制其增殖,同时也下调TGF-β的表达,为进一步研究IL-12的肿瘤治疗作用奠定了基础。Objective：To construct a recombinant adenovirus expression vector carring human IL-12 gene,and investigate its effects on the proliferation and TGF-β expression of Hep3B cells.Methods：The gene of hIL-12 was amplified with PCR and the shuttle plasmid pAdTrack-IL-12 was constructed.The shuttle plasmid was transformed into Adeasier cell after linearization with Pme I for homologous recombination in E.coli BJ5183.After digested with Pac I,the recombinant vector was transfected into AD293 cells to package adenovirus pAd-IL-12.The expression of IL-12 was detected by RT-PCR and Western blot in human hepatoma cell Hep3B infected with pAd-IL-12 virus.The proliferation of Hep3B was evaluated by MTT,and the expression of TGF-β was detected with RT-PCR.Results：The recombinant adenovirus plasmid containing IL-12 was exactly constructed.Recombinant adenovirus pAd-IL-12 were successfully constructed which could infect Hep3B cell efficiently.In Hep3B infected with pAd-IL-12,high expressions of IL-12 mRNA and protein were confirmed by PCR and Western blot,and both of the proliferation and TGF-β expression of Hep3B were significantly reduced（both P0.05） compared with control group.Conclusions： The results demonstrate that recombinant adenovirus containing IL-12 were constructed successfully and IL-12 could suppress Hep3B proliferation with the decline of TGF-β gene,which provides a foundation for further study of IL-12 in tumor treatment.

关 键 词：白介素12 转化生长因子 细胞增殖 肝癌细胞 

分 类 号：R730.5[医药卫生—肿瘤]