胰腺癌细胞总RNA转染树突细胞诱导特异性CTL能力的体外研究

目的观察人胰腺癌Mia Pa Ca-2细胞总RNA电转染树突细胞(Dendritic Cell,DC)体外激发抗原特异性细胞毒T淋巴细胞(Cytotoxic T Lymphocyte,CTL)的能力。方法自6例胰腺癌患者外周血单核细胞中分离、培养DC。使用电穿孔法将Mia Pa Ca-2细胞总RNA体外转录和PCR扩增的MUC1m RNA转染DC,以未负载抗原的DC为对照。采用实时定量PCR技术检测各组DC中MUC1表达。四甲基偶氮唑盐(MTT)检测转染各组DC存活率变化;混合细胞培养法评价各组DC体外刺激自体T淋巴细胞增殖能力;ELISA法检测各组DC体外激发抗原特异性CTL细胞因子释放量。结果 Mia Pa Ca-2总RNA与MUC1 m RNA分别转染后48 h DC中目标抗原的相对表达量分别为37.24±3.17和34.53±2.02,两者比较无显著差异(P0.05)。电转染后96 h Mia Pa Ca-2总RNA转染组DC存活率降至60.81%,低于MUC1 m RNA单转染时DC的存活率(80%左右)(P0.05)。转染Mia Pa Ca-2总RNA DC刺激自体T细胞增殖指数为8 432±611.25,显著高于MUC1单独转染组3 664±305.17(P0.05);且转染Mia Pa Ca-2总RNA DC激发特异性CTL分泌IL-2、IL-10、Granzyme B、IFN-γ水平亦显著高于MUC1 m RNA单独转染组(P0.05)。结论胰腺癌肿瘤细胞总RNA转染的DC较单一胰腺癌相关抗原负载DC有更强的体外抗原特异性CTL激发能力。

The induction of specific cytotoxic T lymphocytes by the dendritic cells transfected with total RNA of ;pancreatic cancer MiaPaCa-2 cells in vitro

Objective To investigate the ability of induction of specific cytotoxic T lymphocytes （CTL） stimulated by dendritic cells （DC） transfected with total RNA of human pancreatic cancer MiaPaCa-2 cell lines. Method DCs were isolated and cultured from peripheral blood mononuclear cells （PBMCs）. Total RNA derived from MiaPaCa-2 cell lines and MUC1 mRNA were transfected into DCs by electroporation respec-tively. The expression of MUC1 in DCs was detected by quantitative real-time PCR. The survival rate of transfected DCs were determined by MTT method. The lymphocyte proliferation ability was evaluated by mixed cell culture method. The cytokines releasing of antigen-specific CTLs were measured by ELISA assay. Results After total RNA of MiaPaCa-2 cell lines and MUC1 mRNA transfection for 48h, the expression of MUC1 were 37.24 ± 3.17 and 34.53 ± 2.02 （P〉0.05）. After MUC1 mRNA was individually transfected, the survival rate of DCs was stabilized around 80%, and the survival rate of DCs was 60.81%96 hours after the total RNA of MiaPaCa-2 cell lines transfection （P〈0.05）. The autologous T cell proliferation index of the to-tal RNA of MiaPaCa-2 cell lines transfection was 8,432 ± 611.25 in DC group, which was significantly higher than the single MUC1 transfection group （ 3,664 ± 305.17） （P〈0.05）. The levels of IL-2, IL-10, Granzyme B, IFN-γsecreted by MUC1 and the total RNA of MiaPaCa-2 cell line transfected DC-specific CTL were significantly higher than MUC1 mRNA alone transfection group （P〈0.05）. Conclusion DCs transfected with the total RNA of pancreatic cancer MiaPaCa-2 cell lines have a stronger ability to stimulate specific CTL in vitro than the single antigen loaded DCs.