胰腺癌MUC1 mRNA转染树突细胞疫苗诱导特异性抗肿瘤的免疫反应  

Induction of specific anti-tumor immune responses against pancreatic cancer by transfected dendritic cells with pancreatic cancer MUC1 mRNA

作　　者：陈江[1] 郭晓钟[1] 李宏宇[1] 邵晓东[1] 刘旭[1] 赵佳钧[1] 王迪[1] 

机构地区：[1]沈阳军区总医院消化科,沈阳110016

出　　处：《中华胰腺病杂志》2012年第3期156-159,共4页Chinese Journal of Pancreatology

基　　金：国家自然科学基金（81071982）

摘　　要：目的研究人胰腺癌MUC1 mRNA转染树突细胞（DC）诱导的特异性抗肿瘤免疫反应，为DC疫苗治疗胰腺癌提供实验依据。方法从外周血中分离单核细胞（PBMC）并培养成DC，从细胞形态和表面标志进行鉴定。通过RT-PCR扩增胰腺癌MiaPaCa-2细胞的MUC1 mRNA后用电穿孔法将其转染DC。采用实时定量PCR和蛋白质印迹法检测培养不同时间点DC的MUC1的表达。用四甲基偶氮唑蓝法检测DC存活率。采用^51Cr标准细胞毒实验观察转染MUC1 mRNA的DC诱导的特异性细胞毒性T淋巴细胞（CTL）反应；应用ELASA法检测CTL的IFN-γ释放量。结果培养获得的细胞呈现典型的DC形态特征和表面标志（CD40^＋、HLA-DR^＋、CD83^＋、CD86^＋）。MUC1mRNA转染DC48h后，细胞MUC1 mRNA表达水平最高，为38．43（36．89～48．06），蛋白表达亦最强。转染后DC的存活率稳定在80％左右。转染MUC1 mRNA的DC可有效诱导HTJA—A2^＋／MUC^＋特异性CTL免疫反应；胰腺癌Capan-2细胞与转染MUC1的DC刺激MUC1特异性CTL的24h IFN-1释放量分别为（28．44±4．96）和（16．31±2．54）U／ml，差异具有统计学意义（P〈0．05）。结论人胰腺癌MUC1 mRNA体外转染DC后可诱导CTL产生特异性抗肿瘤免疫反应。Objective To investigate the induction of specific anti-tumor immune response by transfected dendritic cells （DCs） with MUC1 mRNA of human pancreatic cancer, and to provide the experimental evidences for the treatment of human pancreatic cancer with DC vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells （ PBMCs ） , and then were identified by cell morphology and surface markers. After being transcripted and amplified, MUC1 mRNA was transfected into DCs by electroporation. The expression of MUC1 in DCs at different time points was detected by quantitative real-time PCR and Western blot. The survival rate of DCs before and after transfection was determined by MTT method. The induction of specific cytotoxic T lymphocyte （CTL） response by MUC1 mRNA transfected DCs was measured by ^51 Cr standard cytotoxieity test. The released amount of IFN-γ was evaluated by ELISA method. Results The cultured cells appeared typical characteristics with regard to morphology and phenotype （CD40^＋ , HLA-DR^＋ , CD83^＋ , CD86^＋ ）. After MUC1 mRNA transfection for 48 h, the expression of MUC1 mRNA of DCs reached the highest point （38.43） and the MUC1 protein expression also reached the highest point at 72 h. The survival rate of DCs was stabilized around 80% after transfection. The DCs transfected with MUC1 mRNA could effectively induce HLA-A2^＋/MUC1 ＋ specific CTL immune responses. Stimulated by pancreatic cancer cell line Capan-2 cells or the DCs transfected with MUC1 mRNA, the IFN-γ released in 24 h by MUC1 specific CTL were （ 28.44± 4.96 ） U/ml and （ 16.31± 2.54 ） U/ml, respectively. The difference between the two groups was statistically significant （ P 〈 0.05 ）. Conclusions DCs transfected with human pancreatic cancer MUC1 mRNA could induce CTLs and produce specific anti-tumor immunity.

关 键 词：树突细胞 RNA 信使 转染 胰腺肿瘤 T淋巴细胞 细胞毒性 

分 类 号：R735.9[医药卫生—肿瘤]