慢病毒介导的hGM—CSF修饰的肿瘤疫苗免疫活性研究

目的构建编码人粒细胞-巨噬细胞集落刺激因子（humangranulocyte—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BamHI和XhoI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Lenti—hGM—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％o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。

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

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.