尤文肉瘤融合基因修饰的重组腺病毒的构建及其转染的树突细胞体外抗尤文肉瘤免疫应答  

Construction of recombinant adenoviral vector containing gene of EWS-FLI1 and antitumor immunity of its modified dentritic cell in vitro

作　　者：曲华毅[1] 郭卫[1] 何湘君[2] 

机构地区：[1]北京大学人民医院骨与软组织肿瘤治疗中心,北京100044 [2]北京大学人民医院中心实验室,北京100044

出　　处：《北京大学学报（医学版）》2006年第6期623-627,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30000763)资助~~

摘　　要：目的:构建尤文肉瘤融合基因EWS-FLI1重组腺病毒,利用此腺病毒感染外周血单核细胞(PBMC),培养树突细胞(DC),检测感染后的DC致敏的淋巴细胞对尤文肉瘤细胞系的杀伤作用。方法:将质粒Pec1/EW S-FLI1酶切,切出的EWS-FLI1 cDNA片段克隆至腺病毒穿梭质粒padtrack-cmv的hCMV启动子下游。将穿梭质粒和骨架质粒共同转化大肠杆菌B J5183菌株,获得同源重组后的腺病毒质粒pADeasy-1/EW S-FLI1。将此质粒转染293细胞,包装产生腺病毒Ad EW S-FLI1。扩增、纯化产生高滴度的Ad EW S-FLI1。转染PBMC并经过鉴定后致敏淋巴细胞,4 h51Cr释放试验测定致敏后的淋巴细胞对尤文肉瘤细胞系的杀伤作用。结果:同源重组后产生的pADeasy-1/EW S-FLI1经PCR鉴定构建成功,纯化后的滴度为4×1010/mL。转染PBMC后,RT-PCR证实有EW S-FLI1 mRNA的转录。致敏后的淋巴细胞对尤文肉瘤细胞系有明显的杀伤作用,效靶比在40∶1时,对尤文肉瘤673细胞系的杀伤率为35.1800%±0.0128%,和对照组相比差异有统计学意义。结论:重组腺病毒Ad EW S-FLI1构建成功,并能在PBMC中稳定有效地表达,Ad EW S-FLI1转染的DC可高效地诱发机体T细胞的特异性抗肿瘤免疫应答作用。Objective： To construct an adenoviral vector containing cDNA of EWS-FLI1 and detect its expression in peripheral blood mononeuclear cell（ PBMC ）. To Investigate the antitumor immunity in vitro of the EWS-FLI1 gene modified-dendritic cells. Methods： The EWS-FLI1 cDNA in plasmid Pecl/EWS- FLI1 was digested and subcloned into the shuttle plasimid padtrack-cmv. The shuttle plasmid and the bone plasmid pADeasy-1 were cotransformed into BJ5183 cells. The recombinant plasmid was generated by homologous recombination in BJ5183 cells. The positive clone was obtained by digestion and electrophoresis. Transforming the recombinant plasmid into ＂293 cells＂ by lipofectamine method. Adenoviruses with high titer and purity were obtained by amplifying in the＂293 cells＂ on a large scale and uhra-centrifugation in CsCL step gradient solutions. The cytotoxic activity of stimulated T cells to Ewing sarcoma cells was detected by ^51Cr release assay. Results： PCR showed that the adenovirus contained EWS-FLI1 cDNA. After the PBMC were transfected by Ad EWS-FLI1, the EWS-FLI1 mRNA was detected by RT-PCR. The antigen-specific CTL was induced successfully by the EWS-FLI1 gene modified-DC. The vigorous antigen-specific CTL response against A673 cells was detected by ^51Cr release assay. The killing percentage was 35.18% ±0.0128% at effector-target ratio 40： 1, which was more efficient than that of the control. Conclusion： The recombinant adenovirus was successfully constructed and could efficient express EWS-FLI1 in PBMC. After T lymphocytes were stimulated by DCs modified with EWS-FLI1 gene, the specific CTL response against Ewing＇ s sarcoma cell line A 673 in vitro was observed successfully. ^51Cr release assay showed that there was significant difference between the experimental group and the control group.

关 键 词：腺病毒科 肉瘤 Ewing 重组融合蛋白质类 树突细胞 

分 类 号：R730.262[医药卫生—肿瘤]