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目的:探讨转4-1BBL肿瘤细胞联合抗CD28单抗体外诱导抗肿瘤活性的能力。方法:通过脂质体法将重组载体pEGFP/neo-h4-1BBL转染人舌鳞癌细胞Tca8113,经G418(400μg/mL)筛选及有限稀释后,获得稳定高表达克隆,分别用RT-PCR和Western印迹检测转染细胞中h4-1BBLmRNA和蛋白的表达。将转染与未转染4-1BBL基因的Tca8113细胞用丝裂霉素C(MMC)处理后,制成肿瘤细胞瘤苗。联合抗CD28单克隆抗体与经体外抗CD3mAb诱导的人外周血T淋巴细胞共同培养,测定T细胞增殖、CTL杀伤活性及产生细胞因子(IL-2和IFN-γ)的能力。实验数据以SPSS12.0软件包进行方差分析。结果:h4-1BBL基因真核表达载体在Tca8113细胞中获得稳定表达。转染h4-1BBL基因的Tca8113细胞联合抗CD28单抗能显著刺激T细胞活化、增殖(P<0.01),促进IL-2、IFN-γ分泌(P<0.01),并能有效地诱导CTL的特异性杀伤活性(P<0.01)。结论:转4-1BBL肿瘤细胞联合抗CD28抗体能显著增强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。
Objective: To investigate the ability of anti-tumor activity of transfected 4-1BBL tumor cells combined with anti-CD28 monoclonal antibody in vitro. Methods: The recombinant vector pEGFP / neo-h4-1BBL was transfected into human tongue squamous cell carcinoma cell line Tca8113 by lipofectamine. After screening and limited dilution with G418 (400μg / mL), stable and highly expressed clones were obtained. Western blot was used to detect the expression of h4-1BBL mRNA and protein in transfected cells. The Tca8113 cells transfected and untransfected with 4-1BBL gene were treated with mitomycin C (MMC) to form tumor cell vaccine. The anti-CD28 monoclonal antibody was co-cultured with human peripheral blood T lymphocytes induced by anti-CD3 mAb in vitro to determine the ability of T cell proliferation, CTL killing and production of cytokines (IL-2 and IFN-γ). The experimental data were analyzed by ANOVA with SPSS12.0 software package. Results: The h4-1BBL gene eukaryotic expression vector was stably expressed in Tca8113 cells. Tca8113 cells transfected with h4-1BBL gene combined with anti-CD28 mAb significantly stimulated the activation and proliferation of T cells (P <0.01), promoted the secretion of IL-2 and IFN-γ (P <0.01), and effectively induced CTL Specific killing activity (P <0.01). Conclusion: The combination of 4-1BBL tumor cells with anti-CD28 antibody can significantly enhance the immunogenicity of tumor cells and induce effective anti-tumor immune response of T cells.