双胰腺癌相关抗原RNA转染树突细胞诱导特异性抗癌免疫反应的实验研究  

Induction of specific immune cytotoxic T lymphocyte responses against pancreatic cancer by transfected dendritic cells with MUC4 and Survivin mRNA in vitro

作　　者：陈江[1] 郭晓钟[1] 李宏宇[1] 邵晓东[1] 许文达[1] 

机构地区：[1]沈阳军区总医院消化科,110016

出　　处：《现代消化及介入诊疗》2014年第1期7-12,共6页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：国家自然科学基金(81071982)

摘　　要：目的研究人胰腺癌MUC4与Survivin mRNA联合转染树突细胞(DC)诱导的特异性抗肿瘤免疫反应,为构建负载多抗原表位DC疫苗治疗胰腺癌提供实验依据。方法自胰腺癌患者外周血单核细胞中分离、培养DCs。使用体外转录和胰腺癌PCR技术扩增MUC4和Survivin mRNA后用电穿孔法将其联合转染DC。采用Western blot技术检测DCs中MUC4和Survivin的表达。用四甲基偶氮唑盐(MTT)法检测转染前后DCs存活率变化;使用IFN-γ酶联免疫法检测MUC4 mRNA与Survivin mRNA联合转染后DC诱导的细胞毒性T淋巴细胞(CTL)的活化反应。采用51Cr标准细胞毒实验检测转染MUC4和Survivin mRNA后DCs诱导的特异性CTL对体外胰腺癌细胞的杀伤作用。结果 MUC4与Survivin mRNA联合转染后72 h DCs中两者的相对表达量低于其分别转染。顺序转染后96 h DCs存活率降至50.2%,低于MUC4 mRNA与Survivin mRNA分别转染时DC 80%的存活率(P<0.05)。MUC4和Survivin mRNA联合转染DC诱导的特异性CTL 24 h IFN-γ释放量达(33.84±3.51)U/mL,高于MUC4与Survivin mRNA分别转染DC诱导的CTL IFN-γ释放水平[(21.87±4.12)U/mL和(16.61±2.09)U/mL,P<0.05]。DCs经MUC4 mRNA与Survivin mRNA联合转染后,可有效诱导HLA-A2+/MUC4+/Survivin+特异性CTL免疫反应,对体外培养的胰腺癌细胞具有显著的杀伤作用。结论 MUC4与Survivin mRNA联合转染的DCs可较单胰腺癌相关抗原负载DCs诱导出更加显著的特异性CTL抗肿瘤免疫。Objective To investigate the induction of specific anti-tumor immune response induced by MUC4 and Survivin mRNA co-transfected dendritic cells （DCs） to provide the experimental evidences for the treatment of human pancreatic cancer with multi-epitope loaded DC vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells （PBMCs）. After being transcripted and amplified, MUC4 and Survivin mRNA were co-transfected into DCs by electroporation. The expression of MUC4 and Survivin in DCs were detected by Western blot. The survival rate of transfected DCs were determined by MTT method. The induction of specific CTL activation by MUC4 and Survivin mRNA co-transfected DCs were evaluated through testing released IFN-γ by ELISA method. The induction of specific cytotoxic T lymphocyte （CTL） re-sponse by MUC4 and Survivin mRNA co-transfected DCs were measured by 51Cr standard cytotoxicity test. Results After MUC4 and Survivin mRNA co-transfection for 72 hours, the expression amount of MUC4 and Survivin were lower than the expression amount of MUC4 or Survivin individually. Compared with the MUC4 or Survivin mRNA individual transfected DCs, the IFN-γreleased in 24 hours by CTLs stimulated with MUC4 and Survivin mRNA co-transfection DCs were （33.84 ＆#177; 3.51）U/ml which was significantly higher than the amount of （21.87 ＆#177; 4.12）U/ml by MUC4 or （16.61 ＆#177; 2.09）U/ml by Survivin mRNA individually （P ＆lt; 0.05）. DCs co-transfection with MUC4 and Survivin mRNA could effectively induce HLA-A2 ＋/MUC4＋/Survivin ＋ specific CTL immune responses against pancreatic cancer cells in vitro. Conclusion The induction of CTLs by DCs co-transfected with human pancreatic cancer MUC4 and Survivin mRNA could produce more powerful specific anti-tumor immunity than single antigen loaded DCs.

关 键 词：树突细胞 RNA转染 胰腺肿瘤 细胞毒性T淋巴细胞 

分 类 号：R392.11[医药卫生—免疫学]