目的 观察人胰腺癌Capan-2细胞总RNA转染的树突细胞(DCs)所诱导的抗肿瘤免疫反应.方法 从6例胰腺癌患者外周血单核细胞中分离、培养DCs.使用电穿孔法将Capan-2细胞总RNA及MUC4 mRNA分别转染DCs.应用四甲基偶氮唑蓝(MTT)法检测转染后DCs的存活率.采用蛋白质印迹法检测DCs中MUC4 mRNA的表达.使用IFN-γ酶联免疫法检测DCs诱导的细胞毒T淋巴细胞(CTLs)的活化反应.采用51Cr标准细胞毒实验检测DCs诱导的抗原特异性CTLs对体外胰腺癌细胞的杀伤效应.结果 Capan-2细胞总RNA转染的DCs(DC-Capan-2-total RNA)的存活率呈时间依赖性下降,转染后96 h的存活率降低至60.8％,而转染MUC4 mRNA的DCs(DC-MUC4 mRNA)的存活率稳定在80.0％左右,两转染组DCs的差异具有统计学意义(P＜0.05).DC-Capan-2-total RNA的MUC4蛋白表达量亦显著低于DC-MUC4 mRNA(P＜0.05).DC-Capan-2-total RNA诱导的CTLs 24 h IFN-γ释放量为(89.34±3.85) U/ml,DC-MUC4 mRNA为(21.77±2.14) U/ml,两转染组的差异有统计学意义(P＜0.05).DC-Capan-2-total RNA诱导的特异性CTLs能够有效识别和杀伤HLA-A2 +/MUC4+的Capan-2细胞及HLA-A2 +/MUC4-的PANC1细胞,而不能有效识别和杀伤HLA-A2-/MUC4-的MiaPaCa-2细胞和HLA-A2-/MUC4+的AsPC-1细胞.结论 胰腺癌细胞总RNA转染的DCs较单个胰腺癌相关抗原转染的DCs能诱导出更加显著的CTLs抗肿瘤免疫反应,但受到MHC Ⅰ类抗原递呈的限制.%Objective To investigate the specific anti-tumor immune response induced by dendritic cells (DCs) transfected with total RNA of human pancreatic cancer Capan-2 cells.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) derived from six patients with pancreatic cancer.Total RNA of Capan-2 cells and MUC4 mRNA were transfected into DCs by electroporation.The survival rate of transfected DCs was determined by MTT method and the expression of MUC4 mRNA in DCs was detected by Western blotting.The activity of cytotoxic T lymphocyte cells (CTLs) induced by DCs transfected with total RNA of Capan-2 cells were evaluated by IFN-γ ELISA and the induction of specific CTL response to the killing effect on pancreatic cancer cell in vitro were measured by 51 Cr standard cytotoxicity test.Results The survival rate of DCs transfected with total RNA of Capan-2 cells (DC-Capan-2-total RNA) showed a decrease in a time dependent manner and the survival rate was reduced to 60.81％ after transfection for 96 h.The survival rate of MUC4 mRNA transfection DCs (DC-MUC4 mRNA) was stable at around 80％.The difference of DCs surviral rate between the two groups was statistically significant (P ＜ 0.05).The amount of MUC4 protein expression of DC-Capan-2-total RNA was significantly lower than that of DC-MUC4 mRNA (P ＜0.05).The quantity of CTL IFN-γ release induced by DC-Capan-2-total RNA was (89.34 ± 3.85)U/mL and the quantity of DC-MUC4 mRNA induced CTL IFN-γ release was (21.77 ± 21.77)U/ml There was statistically significant between the two groups (P ＜0.05).In addition,the specific CTLs induced by DC-Capan-2-total RNA could effectively identify and kill the HLA-A2+/MUC4+ Capan-2 and the HLA-A2+/MUC4-PANC 1 cells,and could not effectively identify and kill the HLA-A2 /MUC4-MiaPaCa-2 cells and the HLA-A2-/MUC4 + AsPC-1 cells.Conclusions A more pronounced CTL anti-tumor immune response can be induced by DCs transfected with total RNA of Capan-2 cells compared with a single tumor associated antigen,but it is limited by MHC class Ⅰ antigen presented.
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