胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应  

Dendritic cells expressing muc4 gene induce cytotoxic T-cell responses in vitro

作　　者：任华建[1] 张烨[1] 卫积书[1] 高文涛[1] 徐泽宽[1] 苗毅[1] 

机构地区：[1]南京医科大学第一附属医院普外科,210029

出　　处：《中华实验外科杂志》2009年第7期830-832,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30500492、30471691）

摘　　要：目的构建含有胰腺癌相关抗原黏蛋白（MUC）4（sv12）的腺病毒载体并转染树突状细胞（DC）构建DC疫苗，观察DC疫苗对胰腺癌细胞的免疫效应。方法NCBI获得MUC4（sv12）mRNA序列，利用片段内酶切位点和重叠聚合酶链反应（PCR）技术获得MUCA（sv12）的编码序列，编码序列克隆到腺病毒载体，构建重组腺病毒（rAd—MUC4）。病毒颗粒感染树突状细胞，产生活化的MUCA特异性细胞毒性T淋巴细胞（CTL），乳酸脱氢酶（LDH）释放法和Elispot法分别检测体外杀伤活性及干扰素（IFN）-γ的表达，同时设置空病毒（GFP）组和磷酸盐缓冲液（PBS）组作为阴性对照。结果成功构建含有胰腺癌相关抗原MUC4（sv12）基因的重组腺病毒，LDH法检测结果：MUCA特异性CTL对HLA-A2＋和MUC4＋Capan-1细胞的毒性[E／T=10：1时为（13．7±6．0）％，20：1时为（21．4±4．7）％，40：时为（36．1±9．5）％]，高于HLA—A2＋Mcf-7细胞及MUCA＋Bxpc-3细胞（P〈0．05）。Elispot法检测结果：E／T=20：时，Capan．1细胞的MUCA组斑点数为（139．1±23．3），高于GFP组（66．0±16．4）和PBS组（30．5±4．4），P〈0．05；GFP组和PBS组差异无统计学意义。结论腺病毒介导MUC4基因修饰的DC疫苗，可以体外诱导产生特异性CTL细胞，并获得有效的HLA限制性杀伤效应。Objective To obtain the dendritic cells （DC）-based vaccine modified by adenovirus containing MUCA gene, and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells, Methods The mRNA sequence of tumor associated antigen, MUCA, was obtained from NCBI, and MUCA sequence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus （rAd-MUC4）. The DCs were infected by rAd-MUCA virus and then stimulated the lymph cells from the same donor to induce MUCA specific cytotoxicity T lymphocytes （CTL）. The emcacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-~ release. Results The recombinant adenovirus containing MUC4 （svl2） gene was obtained. The MUC4- induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ （ 13.7 ± 6.0 ） % , （ 21. g ±4.7） %, （36.1 ± 9.5）% at ratios of 10： , 20： ,40： ], higher than MCF-7 and Bxpc-3 ceils respectively, P 〈0.05. The spots number of CTL induced by rAd-MUG4 was （ 139.1 ±23.3） ,more than GFP and PBS control group,P 〈 0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL,which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.

关 键 词：胰腺癌 疫苗 腺病毒载体 树突状细胞 

分 类 号：R735.9[医药卫生—肿瘤] R392.12[医药卫生—临床医学]