HPV 16 E6E7基因修饰树突状细胞疫苗诱导CTL致CaSki细胞凋亡体外实验研究  

Construction of genetically modified dendritic cell vaccine expressing HPV-16 E6E7 fusion gene and activating specitific cytotoxic T lymphocytes to induce apoptosis CaSki cells in vitro

作　　者：焦庆昉[1,2] 易发平[1,3] 汪长东[1,3] 袁成福[1,3] 刘勒[1,3] 兰欢[1,3] 符少月[1,3] 艾青[1,2] 宋方洲[1,3] 

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学免疫学教研室,重庆400016 [3]重庆医科大学生物化学与分子生物学教研室,重庆400016

出　　处：《中国免疫学杂志》2011年第2期115-119,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(30800945);重庆市科委自然科学基金项目(CSTC;2008BB5225)资助

摘　　要：目的:采用人乳头状瘤-16(HPV-16)E6E7重组腺病毒(pAd-E6E7)转染树突状细胞(Dendritic cell,DC),观察基因修饰的DC疫苗诱导细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte,CTL)致使CaSki细胞凋亡的效果。方法:将pAd-E6E7转染体外培养的小鼠未成熟树突状细胞制备DC疫苗,激光共聚焦显微镜观察转染的小鼠未成熟树突状细胞绿色荧光蛋白表达,流式细胞术检测转染前后小鼠树突状细胞表面标志物(CD40、CD86、MHCⅡ和CD11C)。DC疫苗诱导产生特异性细胞毒性T淋巴细胞,与CaSki细胞共培养后,采用DAPI、TUNEL及流式细胞术检测CaSki细胞凋亡情况。结果:pAd-E6E7成功转染体外培养的小鼠未成熟树突状细胞,体外转染效率约为40%～50%,成功制备了HPV16 E6E7基因修饰树突状细胞疫苗,诱导产生细胞毒性T淋巴细胞,经DAPI、TUNEL及流式细胞术检测证明CaSki出现凋亡。结论:以带有HPV16 E6E7基因的重组腺病毒载体转染DC制备基因修饰的DC疫苗,诱导CTL致使CaSki细胞出现凋亡。Objective：To develop the recombinant adenovirus carrying HPV-16 E6E7（pAd-E6E7） to infect mouse immature dendritic cells,and to observe the apoptosis of CaSki cells induced by specific cytotoxic T lymphocytes（CTL） triggered by genetically modified DC vaccine expressing HPV-16 E6E7 fusion gene in vitro.Methods：The pAd-E6E7 was used to infect mouse immature dendritic cells to prepare DC vaccine,the confocal microscopy was used to observe the expression of green fluorescence protein in immature dendritic cells,and flow cytometry（FCM） was used to examine the surface expression of CD40,CD86,MHCⅡ and CD11C in both uninfected and infected dendritic cells.DC vaccine was used to induce specific CTL,and after co-culturing the CTL with CaSki cells,DAPI,TUNEL and flow cytometry（FCM） were used to examine the apoptosis of CaSki cells.Results：Immature mouse dendritic cells were successfully transfected by pAd-E6E7 in vitro,and the transfecting efficiency was 40%-50%.DC vaccine was successfully prepared and used to induce specific CTL.CaSki cells underwent apoptosis via coculturing with the specific CTL detected by DAPI,TUNEL and flow cytometry（FCM）.Conclusion：Genetically modified DC vaccine expressing HPV-16 E6E7 fusion gene could induce CTL,and the CTL could induce apoptosis of CaSki cells in vitro.

关 键 词：人乳头状瘤病毒 细胞毒性T淋巴细胞 CASKI细胞 树突状细胞 

分 类 号：R392.11[医药卫生—免疫学] R737.33[医药卫生—基础医学]