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].

作者信息

You Hong-yu, Lian Wei-guang, Zhang Huan-ling, Wang Jun-xia, Zhang Kai-xia, Song Shu-xia

机构信息

Department of Molecular Biology, Hebei Medical University, Shijiazhuang, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2012 May;34(5):336-40. doi: 10. 3760/cma.j.issn.0253-3766.2012.05.004.

DOI:10. 3760/cma.j.issn.0253-3766.2012.05.004

PMID:22883452

Abstract

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 20 µg/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-bearing mouse models was evaluated. Expression of MHC II, 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.

摘要

目的

探讨顺铂与树突状细胞（DC）疫苗联合应用对荷瘤小鼠的抗肿瘤机制。

方法

将B16黑色素瘤细胞在体外以终浓度20μg/ml的顺铂处理24小时。采用蛋白质免疫印迹法检测高迁移率族蛋白B1（HMGB1）、热休克蛋白70（Hsp70）和转化生长因子-β（TGF-β）的表达。建立B16荷瘤小鼠模型。评估顺铂（100μg/小鼠，腹腔注射，连续3天）与瘤内注射DC细胞（3×10⁶/小鼠，间隔7天注射两次）联合应用对荷瘤小鼠模型的治疗效果。采用流式细胞术分析主要组织相容性复合体II类分子（MHC II）、细胞间黏附分子-1（ICAM-1）和B7-2（CD86）的表达。在接种肿瘤细胞28天后处死小鼠。取出肿瘤并称重，取组织样本进行病理检查。通过不连续梯度离心法分离肿瘤浸润淋巴细胞（TIL）。采用流式细胞术分析TIL中调节性T细胞（T-reg）和CD8⁺T细胞的分布，并测定CD8⁺T/T-reg比值。采用微量细胞毒性试验测定细胞毒性淋巴细胞（CTL）的活性。