慢病毒介导的hGM-CSF修饰的肿瘤疫苗免疫活性研究  

Immunocompetence of dendritic cells transfected with lentiviral vector-encoding human granulocyte-macrophage colony-stimulating factor

作　　者：林阿丽[1] 孙青[2] 

机构地区：[1]杭州市第三人民医院病理科,310009 [2]山东省千佛山医院病理科

出　　处：《浙江医学》2013年第22期1967-1971,共5页Zhejiang Medical Journal

基　　金：山东省科技发展计划项目(2004GG3202003)

摘　　要：目的构建编码人粒细胞-巨噬细胞集落刺激因子(human granulocyte-macrophage colony stimulating factor,hGM-CSF)的慢病毒载体,研究编码hGM-CSF的慢病毒载体对树突状细胞(dendritic cell,DC)疫苗抗肿瘤活性的增强作用。方法以质粒pORFhGM-CSF为模板,采用PCR方法扩增出hGM-CSF基因片段;通过BamH I和Xho I限制性内切酶位点,将hGM-CSF基因定向克隆到慢病毒连接载体L166中构建重组载体L166-hGM-CSF。将重组载体L166-hGM-CSF和包装载体L205以及包膜载体L311共同转染慢病毒包装细胞293T,72h后收集病毒上清液获得编码hGM-CSF的慢病毒颗粒LentihGM-CSF。从脐血中分离出单核细胞,rhGM-CSF、rhIL-4和α-TNF诱导获得DC,通过相差显微镜和流式细胞仪鉴定。应用反复冻融法制备可溶性肿瘤抗原,分别将负载肿瘤抗原的DC疫苗和慢病毒Lenti-hGM-CSF感染的负载肿瘤抗原的DC疫苗与T淋巴细胞共同培养,MTT方法检测并比较两者所诱导的CTL对反应细胞的体外杀伤活性。结果编码hGM-CSF的慢病毒载体L166-hGM-CSF成功构建,获得了编码hGM-CSF的慢病毒。该慢病毒的Hela细胞上清液中hGM-CSF的表达明显高于未感染者。从脐血中分离出的DC,显示其高表达CD80(87.2%)和CD86(92.8%),而单核细胞标志CD14的表达率较低(3.56%)。慢病毒介导的分泌hGM-CSF的树突状细胞肿瘤疫苗刺激同种T淋巴细胞增殖的能力和抗肿瘤活性较普通DC疫苗明显增强(P<0.01)。结论本实验制备的慢病毒Lenti-hGM-CSF感染反应细胞后能够显著增强DC肿瘤疫苗刺激同种T淋巴细胞增殖的能力及其诱导的CTL细胞对肿瘤细胞的杀伤能力,为hGM-CSF修饰的DC肿瘤疫苗的临床应用提供了一定的实验依据。Objective To construct lentiviral vector encoding human granulocyte- macrophage colony-stimulating factor （hGM-CSF） gene and to investigate the immunocompetence of dendritic cell （DC） vaccine transfected with lenti-hGM-CSF. Methods DNA fragment of hGM-CSF was cloned from the plasmid pORFhGM-CSF with PCR and was inserted into the delivery vector L166 at the restriction nuclease sites BamH I and Xho I directionally to construct the recombinant vector L166-hGM-CSF. The recombinant vector L166-hGM-CSF, packing vector L205 and VSVG L311 were co-transfected into lentivirus packaging cell line 293T, and the lentivirus-produced lenti-hGM-CSF was gained from supernatant after 72h. Cord blood was collected to isolate monocytes which were cultured in medium supplemented with rhGM-CSF,rhlL-4 and α-TNF. Suspending cells were collected to analyze their phenotypes by flow cytometer. Tumor lysate was obtained by rapid freeze/thaw exposures. Dendritic cells pulsed with tumor lysate or dendritic cells pulsed with tumor tysate transfected with lenti-hGM-CSF were co-cultured with T lymphocytes. The killing activity of cytotoxic T lymphocytes （CTLs） activated by these DC vaccines were assayed by M-I-r method. Results The recombinant vector L166- hGM-CSF was successfully constructed, and human GM-CSF was presented highly in the supernatant of Hela cells transfected with lenti- hGM-CSF. The cytotoxic effects of DC pulsed with tumor lysate were significantly enhanced after transfected with lenti-hGM-CSF. Conclusion GM-CSF DC vaccine has been successfully prepared by trasfection of hu- man GM-CSF to dendritic cells using the lentiviral vector, which demonstrates an enhanced anti-tumor effects in vitro.

关 键 词：慢病毒载体 粒细胞巨噬细胞集落刺激因子 树突状细胞 肿瘤疫苗 

分 类 号：R392[医药卫生—免疫学]