SLC基因修饰树突状细胞及不同免疫方式抗肿瘤作用探讨  

Recombinant adeno-associated virus encoding secondary lymphoid tissue chemokine(rAAV-SLC) efficiently infected mouse BMDC

作　　者：梁春敏[1] 钟翠平[1] 郑宁[2] 刘彬彬[2] 孙瑞霞[2] 吴欣[2] 刘银坤[2] 叶胜龙[2] 

机构地区：[1]复旦大学医学院人体解剖与组织胚胎学系,上海200032 [2]复旦大学肝癌研究所,上海200032

出　　处：《中国免疫学杂志》2006年第8期721-726,共6页Chinese Journal of Immunology

基　　金：国家863基金资助项目(2001AA217131);复旦大学"985工程"重中之重学科项目;"211工程"肿瘤学科建设项目基金资助;国家自然科学基金项目(30370748)

摘　　要：目的:应用次级淋巴组织趋化因子(SLC)成熟肽基因/重组腺相关病毒(rAAVSLC)转染树突状细胞(DC),探讨一种基因修饰DC的新方法及其抗肿瘤的生物学活性。方法:体外构建rAAVSLC,按MOI为10、50、100与腺病毒(Ad2)协同转染小鼠骨髓来源成熟和未成熟DC,GFP作为成功转染的示踪蛋白。RTPCR在mRNA水平、ELISA在蛋白水平检测SLC表达,Transwell检测转染DC的培养上清对T细胞的趋化作用。瘤苗的在体效应通过C57BL/6J小鼠肿瘤模型验证。皮下接种Hepal6肿瘤细胞后成瘤小鼠分成3组:①生理盐水对照组,瘤内或足垫注射NS;②单纯DC对照组,瘤内或足垫注射未修饰DC;③SLC基因修饰DC组,瘤内或足垫注射SLC基因修饰的DC。每隔7天注射1次,观察各组小鼠肿瘤的生长情况。4周后分离瘤体,用荧光免疫组化检测瘤体内CD4+T细胞、CD8+T细胞浸润情况。结果:rAAVSLC在MOI为100时能成功转染DC,未成熟DC的转染效率显著高于成熟DC,并产生对T细胞有明显趋化生物学活性的SLC。动物实验表明,瘤内注射SLC修饰后DC,肿瘤大小与对照组有明显差异,免疫组化显示瘤体内有较多的CD4+T细胞、CD8+T细胞浸润。足垫注射SLC修饰的DC与对照组相比,抗肿瘤作用无显著差异。结论:rAAVSLC在Ad2的辅助下能有效转染未成熟DC并能表达具有趋化生物学活性的SLC。SLC基因修饰的树突状细胞瘤内注射以其对CD4+T细胞、CD8+T细胞的吸引作用而发挥了明显的抗肿瘤作用,同时也进一步证实用树突状细胞进行肿瘤生物免疫治疗时,不同免疫方式对疗效有明显影响。Objective：To deliver the gene that encodes the mature peptide of secondary lymphoid tissue chemokine（SLC） into mouse bone marrow derived dendritic cell （BMDC） with rAAV （ recombinant adeno-associated virus ）, and to detect its anti-tumor effect. Mcthods：Anti-CD11c antibody conjugated microbeads were used to isolate mouse imBMDCs and mBMDCs. The BMDCs were infected with rAAV-SLC which had been packaged using pAAV helper free system, and with GFP as a tracing protein. The production of SLC was detected by RT-PCR and Sandwich ELISA; its chemotaxis bioactivity was assayed in transwell culture using T lymphocytes as target cells. The anti-tumor effect of SLC gene modified DCs was detected by tumor-bearing animal models. Different DC delivery manners were used, one is to inject DCs directly into tumors; the other is to deliver DCs by footpad injection. Results：The imBMDCs were more efficiently infected than mBMDCs at MOI 100 with Ad co-infecting. SLC peptide secreted by SLC-AAV-DCs was showed to have strong chemotaxis for T lymphocytes. The results showed that the in tumor injection of DC vaccine could attract more CD4^＋ and CD8^＋T lymphocytes into tumor and thus had stronger anti-tumor effect than that delivered by footpad. Conelusion：rAAV-SLC could infect imBMDCs at high efficiency with Ad co-infecting. A new DC vaccine was constructed and demonstrated to have strong anti-tumor effect by the in vivo experiment using tumor-bearing mice.

关 键 词：重组腺相关病毒 骨髓来源树突状细胞 次级淋巴组织趋化因子 

分 类 号：R73-362[医药卫生—肿瘤]