肿瘤射频消融裂解物致敏树突状细胞的抗肿瘤活性研究

Anti-tumor activity of dendritic cells loading in situ tumor lysate antigen proudced by radiofrequency ablation

目的 观察射频消融（RFA）肿瘤原位裂解物负载树突状细胞（DCs）抗肿瘤活性.方法 制备BALB/c小鼠骨髓来源的DCs,对BALB/c小鼠结肠癌（C26）皮下种植瘤进行RFA灭活,将其原位裂解的肿瘤组织反复冻融后取上清,Lowry蛋白定量法定量,以终质量浓度5 mg/L负载培养第5天的DCs（即Ag-DCs）,采用流式细胞仪分析Ag-DCs与未负载抗原的DCs的表面分子[CD11c、人类主要组织相容性复合体（MHC）-Ⅱ、CD80与CD86]表达的差异.建立3组肿瘤治疗与预防模型分别是Ag-DC组（n=5）、DC组（n=5）与磷酸盐缓冲液（PBS）对照组（n=5）,采用ANOVA方差分析比较3组间种植瘤体积的变化趋势,Log-rank检验比较3组小鼠的生存时间.结果 Ag-DCs的CD80、CD86、MHC-Ⅱ、CD11c表达率分别为86.3％、29.4％、75.4％、82.5％;未处理的DCs表面分子的CD80、CD86、MHC-Ⅱ、CD11c表达率分别为68.6％、15.1％、64.5％、66.7％;小鼠肿瘤预防模型提示7d成瘤率分别为：Ag-DC组60％（3/5）、DC组80％（4/5）、PBS对照组100％（5/5）;且Ag-DC组小鼠的肿瘤体积生长缓慢,与PBS和DC组的免疫小鼠比较,差异有统计学意义（P＜0.01）.治疗模型提示：Ag-DC组小鼠的平均生存期为（39±8）d,DC组小鼠的平均生存期为（28±4）d,PBS对照组小鼠的平均生存期为（22±3）d,差异有统计学意义（P＜0.01）;且Ag-DC组小鼠的肿瘤体积生长缓慢,与PBS和DC组的免疫小鼠比较,差异有统计学意义（P＜0.01）.结论 肿瘤射频消融原位裂解物作为抗原可有效刺激DC产生特异性抗肿瘤免疫效应.

Objective To evaluate the anti-tumor activity of dendritic cells （DCs） loading in situ tumor lysate antigen proudced by radiofrequency ablation （RFA）.Methods DCs derived from bone marrow were prepared,and experimental model of murine colon carcinoma was established for RFA.The supernatant of tumor tissue in situ lysis after repeated freezing and thawing was tested by Lowry protein quantitative statutory,amount to a final concentration of 5 mg/L,then load to the first 5 days of culture DCs （Ag-DCs）,using flow cytometry analysis the differences of the surface molecules [CD11 c,major histocompatibility complex （MHC）-Ⅱ,CD80,CD86] between Ag-DCs and DCs.Three groups of cancer treatment and prevention models were established respectively：Ag-DC group,DC group and PBS control group.Using ANOVA analysis of variance comparing three groups implanted tumor volume trends,Log-rank test to compare three groups of mice survival time.Results The surface of Ag-DCs expressed CD80,CD86,MHC-Ⅱ,CD11c,respectively,86.3％,29.4％,75.4％,82.5％ ; untreated DCs expressed costimulatory molecules CD80,CD86,MHC-Ⅱ,CD11c respectively,68.6％,15.1％,64.5％,66.7％.The cancer prevention model of mice tips seven days tumor grow probabilities：Ag-DC group 60％ （3/5）,DC group 80％ （4/5）,PBS control group 100％ （5/5） ; and Ag-DC mice showed slow-growing tumor volume compared with PBS and DC groups immunized mice,the difference was statistically significant （P ＜ 0.01）.Treatment model suggested：the average survival time of Ag-DC group was （39 ± 8） d,DC group was （28 ± 4） d,PBS group was （22 ± 3） d,the difference was statistically significant （P ＜ 0.01） ; and the growth of tumor volume of Ag-DC group was slow,compared with PBS and DC groups,the difference was statistically significant （P ＜ 0.01）.Conclusion Radiofrequency ablation in situ tumor lysate as an antigen can effectively stimulate DCs to produce specific anti-tumor immune response,suggesting that the antigen loading DCs as an anti-tunor immunotherapy strategie are suitable for clinical application.