CAJ | 학술논문

目的：观察人胰腺癌MiaPaCa-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MiaPaCa-2细胞总RNA体外转录和PCR扩增的MUC1 mRNA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 MiaPaCa-2总RNA与MUC1 mRNA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（P>0.05）。电转染后96 h MiaPaCa-2总RNA转染组DC存活率降至60.81%，低于MUC1 mRNA单转染时DC的存活率（80%左右）（P<0.05）。转染MiaPaCa-2总RNA DC刺激自体T细胞增殖指数为8432±611.25，显著高于MUC1单独转染组3664±305.17（P<0.05）；且转染MiaPaCa-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 mRNA单独转染组（P<0.05）。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。
목적：관찰인이선암MiaPaCa-2세포총RNA전전염수돌세포（Dendritic Cell，DC）체외격발항원특이성세포독T림파세포（Cytotoxic T Lymphocyte, CTL）적능력。방법자6례이선암환자외주혈단핵세포중분리、배양DC。사용전천공법장MiaPaCa-2세포총RNA체외전록화PCR확증적MUC1 mRNA전염DC，이미부재항원적DC위대조。채용실시정량PCR기술검측각조DC중MUC1표체。사갑기우담서염（MTT）검측전염각조DC존활솔변화；혼합세포배양법평개각조DC체외자격자체T림파세포증식능력；ELISA법검측각조DC체외격발항원특이성CTL세포인자석방량。결과 MiaPaCa-2총RNA여MUC1 mRNA분별전염후48 h DC중목표항원적상대표체량분별위37.24±3.17화34.53±2.02，량자비교무현저차이（P>0.05）。전전염후96 h MiaPaCa-2총RNA전염조DC존활솔강지60.81%，저우MUC1 mRNA단전염시DC적존활솔（80%좌우）（P<0.05）。전염MiaPaCa-2총RNA DC자격자체T세포증식지수위8432±611.25，현저고우MUC1단독전염조3664±305.17（P<0.05）；차전염MiaPaCa-2총RNA DC격발특이성 CTL분비IL-2、IL-10、Granzyme B、IFN-γ수평역현저고우MUC1 mRNA단독전염조（P<0.05）。결론이선암종류세포총RNA전염적DC교단일이선암상관항원부재DC유경강적체외항원특이성CTL격발능력。
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.