人4-1BB配体基因真核表达载体的构建、表达及其体外抗肿瘤效应  

Construction and expression of eukaryotic expression vector of human 4-1BB ligand gene in tumor cells and its antitumor activity in vitro

作　　者：吕坤[1] 吴俊英[1] 李柏青[1] 

机构地区：[1]蚌埠医学院免疫学教研室,安徽蚌埠233030

出　　处：《细胞与分子免疫学杂志》2007年第6期511-514,519,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：安徽省教育厅自然科学研究计划项目(2004kj280;KJ2007A097)

摘　　要：目的:构建人4-1BB配体(h4-1BBL)全长基因的真核表达载体,并在肿瘤细胞HT-29中转染表达;探讨人4-1BBL基因转染的肿瘤细胞体外诱导的抗肿瘤活性。方法:用RT-PCR从Raji细胞中克隆h4-1BBL全长基因,测序后,构建重组真核表达载体pcDNA3.1(-)-h4-1BBL。通过脂质体法以重组载体转染HT-29细胞,用RT-PCR检测转染细胞中h4-1BBLmRNA的表达;用流式细胞术检测转染细胞表面h4-1BBL分子的表达。分离外周血单个核细胞(PBMC),用抗CD3mAb扩增T细胞,并与h4-1BBL基因转染及未转染的HT-29细胞混合培养。用MTT比色法检测CTL的增殖及杀伤活性;用流式细胞术检测分泌IFN-γ的T细胞。结果:从Raji细胞中克隆到h4-1BBL全长cDNA,测序完全正确。构建的h4-1BBL基因真核表达载体在HT-29中获得稳定表达。与未转染的细胞相比较,h4-1BBL基因转染的肿瘤细胞HT-29能更有效地刺激T细胞活化、增殖,促进IFN-γ分泌,并能有效地诱导CTL产生针对野生型HT-29细胞的特异性杀伤。结论:成功地构建pcDNA3.1(-)h4-1BBL重组真核表达载体。4-1BBL基因转染的肿瘤细胞介导的协同刺激信号,能增强野生型肿瘤细胞的免疫原性,诱导T细胞产生有效的抗肿瘤免疫应答。AIM： To construct eukaryotic expression vector of human 4-1BB ligand （4-1BBL） gene and express it in HT-29 cell line. To explore the effect on activation and cytotoxicity of human cytotoxic T lymphocytes （CTLs） induced by human 4-1BBL gene transfection into tumor cells in vitro. METHODS： RT-PCR was applied to amplify the fulllength of human 4-1BBL gene from Raji cells. After sequen- cing, the cDNA was recombinated into the eukaryotic ex- pression vector pcDNA3. 1（-） and transfected into HT-29 cells through Lipofect AMINE 2000. Human 4-IBBL mRNA and protein expression of transfected cells was detected by RT-PCR and FACS respectively. Human peripheral blood mononuclear cells were stimulated with anti-CD3 mAb and incubated with non-transfected or transfected HT-29 cells, respectively. The MTT colorimetry was used to detect the proliferation and cytotoxic effect of T lymphocytes. Meanwhile, the expression of intracellular IFN-γ was detected by FCM. RESULTS： The HT-29 cells transfected by pcDNA3. 1 （-）- h4-1BBL could express human 4-1BBL efficiently. As compared with wild type HT-29 cells, the transfected HT-29 cells had more effect for proliferation, IFN-γ production and cytotoxic activity of lymphocytes. CONCLUSION： The recombinant eukaryotic expression vector of pcDNA3. 1 （-）-h4-1BBL is successfully constructed. The transfection of human 4-1 BBL gene in HT-29 cells is effctive in enhancing its immunogenicity and inducing antitumor immune response in vitro.

关 键 词：h4-1BBL 基因转染 抗肿瘤免疫应答 

分 类 号：R730.3[医药卫生—肿瘤]