EBV-LMP2A重组腺病毒体外转染树突状细胞激发特异性CTL的研究  

Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro

作　　者：姚堃[1] 彭光勇[1] 丁传林[1] 周锋[1] 谢芳艺[2] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]南京医科大学微生物学与免疫学系,南京210029

出　　处：《中国免疫学杂志》2004年第12期816-820,共5页Chinese Journal of Immunology

基　　金：本课题受国家自然科学基金 (批准号 :3 0 170 880 ) ;江苏省科委"九五"重大课题基金 (批准号 :BJ9810 0 )资助

摘　　要：目的 :用EBV潜伏膜蛋白 2A(EBV LMP2A)重组腺病毒转染树突状细胞 (DC)激发特异性细胞毒性T细胞(CTL) ,分析CTL的特性。方法 :用Ad5 LMP2A重组腺病毒转染EBV健康携带者及鼻咽癌患者的DC ,与自体来源的外周血单个核细胞 (PBMC)混合培养 ,激发LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS)检测培养细胞群体中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 细胞的组成 ;生物活性法检测细胞培养上清中IFN γ含量 ;RT PCR分析CTL的FasLmRNA表达。结果 :EBV健康携带者及鼻咽癌患者的PBMC ,经Ad5 LMP2A转染的自体DC两次刺激后 ,都能诱导出显著的EBV LMP2A特异的CTL。EBV健康携带者CTL的杀伤活性 ,随DC刺激次数的增加而逐渐增强 ,诱导的CTL细胞群体以CD4 +和CD8+ 细胞组成为主 ,且CD4 + 细胞比例高于CD8+ 细胞 ,另含少量CD5 6 + 细胞 ;在不同时段所诱导的CTL上清中 ,均含一定量的IFN γ ,并随刺激次数和诱导时间的延长呈上升趋势 ;RT PCR研究表明 ,所诱导的CTL有FasLmRNA的表达。结论 :以腺病毒载体介导EBV LMP2A基因转染的成熟DC ,在体外能激发较强的LMP2A特异的功能性CTL ,可用于EBV相关NPC的免疫治疗。Objective:Generation and functional analysis of EBV LMP2A specific CTL elicited by DC transfected with recombinant adenovirus in vitro .Methods:PBMC were isolated from healthy EBV carriers and NPC patients, and then cocultured with autologous mature Ad5 LMP2A transfected DC at the ratios of 20∶1. Cytotoxicity of LMP2A specific CTL was determined with LDH release assay, the populations of CTL were performed by FACS,the IFN γ secretion and FasL mRNA expression of the CTL were detected by biological activity assays and RT PCR, respectively.Results:The results showed that high cytotoxicity of LMP2A specific CTL could be elicited by autologous transfected DC. The cytotoxicity boosted with the increase of transfected DC stimulation times, but there were no significant changes between two and three stimulations.The phenotypic analysis demonstrated that the LMP2A specific CTL induced at day 14 consisted of a majority of CD4 + and CD8 +T cells, and only a small percentage of CD56 + cells. The IFN γ secreted in the supernatants of cell culture also increased with the stimulation times. In addition, the specific CTL at day 14 from EBV healthy carriers could express FasL mRNA.Conclusion:Strong and functional EBV specific CTL could be generated by autologous mature DC transfected with adenovirus encoding LMP2A, which could provide a rationale for the immunotherapy of EBV associated NPC. [

关 键 词：腺病毒载体 树突状细胞 EB病毒 潜伏期膜蛋白2A 细胞毒性T细胞 

分 类 号：R392.11[医药卫生—免疫学]