咪喹莫特(imiquimod)联合树突状细胞降低荷黑素瘤小鼠调节性T细胞并增强抗肿瘤效果  

Imiquimod combined with dendritic cell vaccine decreases Treg proportion and enhances anti-tumor responses in mice bearing melanoma

作　　者：任书荣[1] 王秋波[1] 张艳丽[1] 路翠秀 李平[1] 李玉美[1] REN Shurong WANG Qiubo ZHANG Yanli LU Cuixiu LI Ping LI Yumei(Department of Immunology, Basic Medical College, Qingdao University, Qingdao 266071, China)

机构地区：[1]青岛大学医学部基础医学院免疫学系,山东青岛266071

出　　处：《细胞与分子免疫学杂志》2017年第2期145-150,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省自然科学基金(ZR2014HM025);山东省医药卫生科技发展计划项目(2014WS0445);青岛大学大学生创新创业训练项目

摘　　要：目的观察联合应用Toll样受体7(TLR7)激动剂咪喹莫特(imiquimod)和负载肿瘤相关抗原的树突状细胞(DC)疫苗对荷黑素瘤小鼠的治疗作用并探讨相关机制。方法培养表达卵白蛋白的B16黑素瘤(B16-OVA)细胞,注射入C57BL/6小鼠皮下制备荷瘤小鼠模型。含重组小鼠粒细胞巨噬细胞集落刺激因子(rm GM-CSF)和重组小鼠白细胞介素4(rm IL-4)的培养液扩增培养骨髓来源的DC,加入OVA过夜孵育制备负载OVA的DC疫苗(OVA-DC)。荷瘤小鼠分别采用PBS、咪喹莫特局部涂抹、OVA-DC皮内注射、咪喹莫特联合OVA-DC进行治疗,数字游标卡尺测量小鼠皮下肿瘤的生长情况。流式细胞术检测荷瘤小鼠外周血CD4^+FOXP3^+调节性T细胞(Treg)的比例。采用上述方案免疫小鼠后,用CCK-8法检测小鼠脾脏淋巴细胞对B16-OVA细胞的杀伤效应。结果与单纯咪喹莫特、OVA-DC治疗组及PBS对照组相比,咪喹莫特联合OVA-DC治疗组荷瘤小鼠黑素瘤体积较小,荷瘤小鼠CD4^+细胞中FOXP3^+Treg比例明显降低。咪喹莫特联合OVA-DC免疫小鼠脾脏淋巴细胞杀伤B16-OVA肿瘤细胞能力较其他组明显增强。结论咪喹莫特联合负载抗原的DC疫苗可诱导荷瘤机体CD4^+FOXP3^+Treg比例降低并增强抗肿瘤效果。Objective To investigate the therapeutic effect of Toll-like receptor 7（ TLR7） agonist imiquimod combined with dendritic cell（ DC）-based tumor vaccine on melanoma in mice and the potential mechanism. Methods Melanoma-bearing mouse models were established by subcutanous injection of B16-OVA cells into C57 BL /6 mice. DCs were isolated from mouse bone marrow and propagated in culture medium with recombinant mouse granulocyte-macrophage colony-stimulating factor（ rm GM-CSF） and recombinant mouse interleukin-4（ rm IL-4）. DC vaccine（ OVA-DC） was prepared by overnight incubation of DCs added with chicken ovalbumin. C57 BL /6 mice were separated into four groups which were treated with PBS,topical imiquimod application,OVA-DC intradermal injection and imiquimod plus OVA-DC,respectively. The tumor size was calculated by digital vernier caliper. Peripheral blood CD4^＋FOXP3^＋Tregs of the tumor-bearing mice was detected by flow cytometry. The cytotoxicity of splenic lymphocyte against B16-OVA was assessed in vitro by CCK-8 assay. Results Compared with the other three groups,B16-OVA-bearing mice treated with imiquimod plus DC vaccine had the smallest tumor volume. The percentage of CD4^＋FOXP3^＋Tregs decreased significantly in the combined treated mice. The combined treatment enhanced significantly cytotoxicity of splenic lymphocytes against B16-OVA cells. Conclusion Imiquimod combined with antigen-pulsed-DC vaccine could reduce CD4^＋FOXP3^＋Treg proportion and promote anti-tumor effect in mice with melanoma.

关 键 词：Toll样受体7(TLR7) 咪喹莫特(imiquimod) 树突状细胞 肿瘤免疫治疗 调节性T细胞 

分 类 号：R392.12[医药卫生—免疫学] R739.5[医药卫生—基础医学]