树突状细胞疫苗联合Imiquimod对小鼠乳腺肿瘤的治疗作用

Treatment of murine mammary carcinoma by dendritic cell vaccine combined with topical application of Imiquimod

目的观察局部应用TLR7激动剂Imiquimod对树突状细胞(dendritic cell,DC)疫苗治疗小鼠肿瘤的影响,寻找增强DC肿瘤疫苗疗效的佐剂。方法培养乳腺肿瘤细胞株EMT-6,反复冻融法制备肿瘤相关抗原,小鼠乳腺区注射EMT-6制备荷瘤动物模型。用含重组小鼠GM-CSF和IL-4的培养液扩增培养Balb/c小鼠来源的BM-DC,负载肿瘤相关抗原(Ag-DC)后进行肿瘤治疗实验。荷瘤小鼠分别采用Imiquimod局部涂抹、Ag-DC皮内注射、Imiquimod联合Ag-DC进行治疗,检测不同处理方法小鼠的肿瘤生长情况。采用上述方案免疫Balb/c小鼠后细胞增殖抑制实验检测小鼠脾脏淋巴细胞对EMT-6细胞的杀伤效应。流式细胞术检测Imiquimod处理局部皮肤对BM-DC向局部引流淋巴结(DLN)迁移的影响。结果 Imiquimod联合Ag-DC治疗荷瘤小鼠可明显抑制肿瘤生长,与单纯Imiquimod、Ag-DC治疗组及NS处理对照组比较差异具有显著性。Imiquimod联合Ag-DC免疫小鼠后脾脏淋巴细胞杀伤肿瘤细胞能力较其它组明显增强。局部涂抹Imiquimod后注射BM-DC,24 h后DLN内BM-DC数量较对照组明显增多。结论 TLR7激动剂Imiquimod可增强BM-DC疫苗诱导的免疫应答,提高肿瘤治疗效果。其机制可能与Imiquimod诱导DLN BM-DC细胞数量增多有关。

The present study was designed to investigate the therapeutic effect of topical application of Imiquimod combined with dendritic cell （DC） vaccine on routine mammary carcinoma. DC was prepared from the bone marrow of BALB/c mice by culturing progenitor cells in routine GM-CSF and IL-4 media. DC was pulsed with EMT-6 lysate for EMT-6 bearing mice therapy and for specific cytotoxic T lymphocyte （CTL） induction. BALB/c mice were divided randomly into four groups which were treated twice with Imiquimod topical application, Ag-DC intradermal injection, Imiquimod in combination with Ag-DC or normal saline, respectively. EMT-6 proliferating inhibition by CTL was assessed in vitro by cck-8 kit; the effect of Imiquimod on BM-DC migration to DLN was evaluated by FACS analysis of DLN ceils. Data showed that topical application of Imiquimod induced BM-DC homing to DLN. Ag-DC combined with Imiquimod usage induced significant higher specific cytotoxic T lymphocyte directed against EMT-6 cells. The combination treatment mice displayed the smallest tumor volumes compared with the other three groups. Taken together, this experiment proves that TLR7 agonist Imiquimod could enhance BM-DC mediated anti-tumor immune response, which may be related with increased number of BM-DC homing to DLN.