黏蛋白4mRNA与人端粒酶反转录酶mRNA联合转染树突状细胞诱导抗胰腺癌免疫反应的体外研究  

Study on the immune responses against pancreatic cancer induced by mucin 4 and human telomerase reverse transcriptase mRNA co-transfected dendritic cells in vitro

作　　者：陈江[1] 郭晓钟[1] 胡志刚[1] 李宏宇[1] 王迪[1] 赵佳钧[1] 

机构地区：[1]解放军沈阳军区总医院消化科,沈阳110840

出　　处：《中华消化杂志》2013年第11期750-755,共6页Chinese Journal of Digestion

基　　金：国家自然科学基金（81071982）

摘　　要：目的研究人胰腺癌黏蛋白4mRNA与人端粒酶反转录酶（hTERT）mRNA联合转染树突状细胞（DC）诱导的抗肿瘤免疫反应，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6例HLA—A2’的胰腺癌患者外周血单个核细胞中分离、培养DC。体外转录和扩增黏蛋白4和hTERTmRNA，电穿孔法将两者分别及联合转染Dc，培养48h。采用实时定量PCR和Western印迹技术检测13（2中黏蛋白4和hTERT的表达。用MTT法监测转染前后DC存活率；使用IFN-7释放试验（EI．IsA法）检测黏蛋白4mRNA和（或）hTERTmRNA转染后Dc诱导的CTL的活化反应。采用51Cr标准细胞毒实验检测转染黏蛋白4mRNA和（或）hTERTmRNA后DC诱导的CTI。对体外胰腺癌细胞株MiaPaCa-2、Capan2、AsPC-1和Panc1细胞的杀伤作用。采用studenl，检验进行统计学处理。Objective To investigate the anti-tumor immune response induced by human pancreatic cancer mucin 4 mRNA and human telomerase reverse transcriptase （hTERT） mRNA eotransfeeted dendritic cells （DC）, and to provide the experimental evidences for the treatment of pancreatic cancer with multi-epitope loaded DC vaccine. Methods DC were isolated from peripheral blood mononuclear cells （PBMC） of six patients with HLA A2~ pancreatic cancer and cultured. Mucin 4 mRNA and hTERT mRNA were transcripted and amplified in vitro, which were transfected into DC separately or in order by electroporation. DC were cultured for 48 hours. The expressions of mucin 4 and hTERT in DC were detected by quantitative real-time polymerase chain reaction （PCR） and Western blot. The survival rates of transfected DC were determined by methyhhiazolyl tetrazolium （MTT） method. The cytotoxic T lymphocyte （CTL） activation induced by mucin 4 mRNA and hTERT rnRNA transfected DC were evaluated by interferon （IFN）-）＇ release assays （enzyme linked immunosorbent assay） method. The cytotoxicity of CTL induced by mucin 4 mRNA and hTERT mRNA transfected DC in pancreatic cancer cell lines MiaPaCa-2, Capan-2, AsPC-1 and Panc-1 was measured by olCr standard cytotoxicity test. Student t test was performed for statistical analysis. Results After in order transfection of mucin 4 mRNA and hTERT mRNA for 48 h, the relative quantity of the expression of mucin 4 and hTERT in DC were 30.09±5.24 and 12.87±3.36, which were lower than the relative quantity of the expression in DC transfected separately （38.54± 6.21 and 36.35±5.03, t=3.469, 6. 721, both P〈0. 05 ）. After transfected in order for 96 hours, the survival rate of DC decreased to 52.17%, which was lower than that of DC transfected separately （around 80~）. The quantity of IFN-7 releasing of specific CTL induced by mucin 4 mRNA and hTERT mRNA cotransfected DC was （32.57±2.01） U/mL in 24 hours, which was higher than that of CTL induced by DC transfected with muci

关 键 词：树突细胞 黏蛋白类 端粒 末端转移酶 RNA 信使 胰腺肿瘤 T淋巴细胞 细胞毒性 

分 类 号：R735.9[医药卫生—肿瘤]