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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 observe the therapeutic effect of imiquimod (Toll-like receptor 7) agonist imiquimod and tumor-associated antigen-loaded dendritic cell (DC) vaccine on mice bearing melanoma and to explore related mechanisms. Methods B16 melanoma cells (B16-OVA) expressing ovalbumin were cultured and injected into C57BL / 6 mice to establish a tumor-bearing mouse model. Bone marrow-derived DCs were expanded by culture medium containing recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4), and incubated with OVA for overnight incubation to prepare OVA DC vaccine (OVA-DC). Tumor-bearing mice were treated with PBS, imiquimod topical injection, OVA-DC intradermal injection, imiquimod combined with OVA-DC, and the growth of subcutaneous tumors was measured by digital vernier caliper. The proportion of CD4 ~ + FOXP3 ~ + regulatory T cells (Tregs) in peripheral blood of tumor-bearing mice was detected by flow cytometry. After the mice were immunized with the above protocol, the killing effect of mouse spleen lymphocytes on B16-OVA cells was detected by CCK-8 assay. Results Compared with simple imiquimod, OVA-DC treatment group and PBS control group, the tumor volume of tumor-bearing mice with imiquimod plus OVA-DC was smaller, and the tumor-bearing mice CD4 ~ + cells FOXP3 ~ + Treg ratio was significantly reduced. The ability of splenic lymphocytes of imiquimod combined with OVA-DC to kill B16-OVA tumor cells was significantly increased compared with other groups. Conclusion Imiquimod combined with antigen-loaded DC vaccine can reduce the proportion of CD4 ~ + FOXP3 ~ + Tregs in tumor-bearing and enhance the anti-tumor effect.