顺铂联合树突状细胞疫苗对荷瘤小鼠的抗肿瘤作用

Anti-tumor effect of cisplatin combined with DC vaccine on tumor-bearing mice

目的探讨顺铂联合树突状细胞（DC）疫苗对荷瘤小鼠抗肿瘤作用的机制。方法用终浓度20μg/ml顺铂诱导体外培养的小鼠黑色素瘤B16细胞凋亡，提取凋亡细胞总蛋白。Westernblot法检测高迁移率蛋白B1（HMGB1）、热休克蛋白70（HspT0）和转化生长因子13（TGF-B）蛋白表达，流式细胞仪分析DCs表面主要组织相容性复合体Ⅱ类（MHCII）、细胞间黏附分子1（ICAM-1）和CD86的表达。制备荷瘤小鼠模型，腹腔注射顺铂（100μg/只），瘤内注射DC疫苗（3×10^6个／只），28d后处死荷瘤小鼠，分离肿瘤组织并称重。采用不连续梯度离心法分离肿瘤浸润淋巴细胞，流式细胞仪检测调节性T细胞（T—reg）和CD8＋T细胞的分布，采用微量细胞毒方法检测细胞毒性T淋巴细胞（CTL）活性。结果顺铂可明显提高B16细胞HMGB1的表达水平。经顺铂诱导的B16凋亡细胞总蛋白负载DC细胞后，其MHCII、CD86＋和ICAM-1的表达率分别为（47．5±8．8）％、（36．2．4-9．2）％和（35．5±8．3）％。在肿瘤移植的第28天，对照组瘤重为（2．1±0．6）g，顺铂组和顺铂联合DC疫苗组瘤重分别为（0．34-0．2）g和（0．5±0．2）g，顺铂组和顺铂联合DC疫苗组瘤重明显小于对照组（P〈0．01）。顺铂联合DC疫苗不仅提高了肿瘤组织中CD8＋T／CD4＋Foxp3＋T细胞比值，还增强了CTL活性。在效靶比为20：1、10：1和5：1时，顺铂联合DC疫苗组荷瘤小鼠的CTL活性分别为（25．0±5．0）％、（22．0±6．0）％和（14．0±4．0）％，显著高于对照组的（8．2±3．6）％、（6．7±1．8）％和（3．6±1．9）％（均P〈0．01）。结论顺铂联合DC疫苗可下调肿瘤微环境的T—reg细胞，提高CTL活性协同发挥抗肿瘤作用。

Objective To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice. Methods B16 melanoma cells were treated with cisplatin at the final concentration of 20μg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-β were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin （100 μg/mouse i. p. , for sequential 3 days） and intratumoral injection of DC cells （3 ×10^6/ mouse, twice with a 7-day interval） in the tumor-beating mouse models was evaluated. Expression of MHC 11 , ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination Tumor infiltrating lymphocytes （TIL） were isolated by discontinuous gradient centrifugation The distribution of T-reg and CD8 ＋ T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8＋ T/T-reg was determined. The activity of cytotoxic lymphocytes （CTL） was determined by microcytotoxicity assay. Results Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC Ⅱ , ICAM-1 and CD86 on DC cells were （47.5 ±8.8） %, （35.5 ± 8.3） % and （36.2 ± 9.2） %, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was （ 2.1 ± 0.6 ） g, that of the cisplatin group was （ 0.3 ± 0.2 ） g and that of cisplatin ＋ DC vaccine group was （0.5 ± 0.2） g, showing a significant inhibition of tumor growth （ P 〈 0.01 ）. Furthermore, the CD8 ＋ T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin ＋ DC vaccine. When the effector-to-target ratio was 20： 1, 10：1 and 5： 1, the CTL activity in the cisplatin ＋ DC vaccine treated mice was （25.0 ±5.0） %, （22.0 ± 6.0） % and （ 14.0 ±4.0） %, respectively, significantly higher than （ 8.2± 3.6） %, （6.7± 1.8 ） % and （3.6 ± 1.9） %, respectively, in the control group （ all P 〈 0.01 ）. Conclusion Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.