免疫健全小鼠胰腺癌肝脏转移模型的建立及免疫环境变化

摘要

Objective To establish the liver metastasis of pancreatic cancer model in immunocompetent mice and observe the changes of systemic and local immune environment.Methods 2 × 106 C57BL/6J mouse syngeneic PancO2 pancreatic cancer cells were injected into the spleen to establish this model.The FCM (fluorescence activated cytometry) was adopted to detect 14 immune cell populations in both the peripheral blood and the metastatic lesions.The real-time RT-PCR was adopted to detect the T helper 1 (Th1) and T helper 2 (Th2) cytokines in metastatic lesions and paratumoral tissues.Results Four weeks after injection, the liver metastasis rate was 100％.Compared to those in normal mice, peripheral T lymphocytes [(31.0±8.1)％ vs.(11.5 ±5.5)％, P＜0.05] and B lymphocytes [(45.4 ±8.2)％ vs.14.3± 6.4) ％, P ＜ 0.05] in the tumor bearing mice were significantly decreased;however, the granulocytes [(22.4 ± 7.8) ％ vs.(71.5 ± 20.3) ％), P ＜ 0.05] and myeloid derived suppressor cells (MDSC) [(5.4 ± 2.5)％ vs.(57.5 ± 18.8)％, P ＜ 0.05] and M2 polarized tumor-associated macrophages (TAM) [(1.5 ± 1.2) ％ vs.(5.6 ± 3.7) ％, P ＜ 0.05] were significantly increased.In the metastatic lesions massive leukocytes (34.7 ± 11.6)％ were infiltrated.The most populations were granulocytes (47.7 ±12.2)％, MDSC (37.4 ± 10.6)％ and M2 polarized TAM (15.2 ±7.4)％ , the dendritic cells (17.6 ± 8.2)％ were less, and the T lymphocytes and B lymphocytes were only (10.6 ± 5.5)％ and (7.8 ± 2.9) ％, respectively.The main cytokine pattern in the metastatic lesions was a marked Th2 type.Conclusion Pancreatic cancer with liver metastasis can induce extensive systemic and local immunosuppression, and MDSC and M2 polarized TAM may play crucial roles.Immune environment reconstruction could be the potential important target for preventing and treating liver metastasis of pancreatic cancer.%目的 建立免疫健全小鼠胰腺癌肝转移模型并观察全身及肿瘤局部免疫环境的变化.方法 利用2×106个C57 BL/6J小鼠同源Panc02胰腺癌细胞脾内注射的方法建立胰腺癌肝转移模型.流式细胞计数检测外周血及肝转移灶组织中14个免疫细胞群的变化.荧光实时定量PCR检测肝转移灶以及癌旁组织辅助T细胞1型(T helper cell 1)、辅助T细胞2型(T helper cell 2)细胞因子的差异表达.结果 建模4周后,肝转移率为100％.与正常对照小鼠相比,胰腺癌肝转移小鼠外周血T淋巴细胞[(31.0±8.1)％比(11.5±5.5)％,P ＜0.05]以及B淋巴细胞[(45.4±8.2)％比(14.3±6.4)％,P＜0.05]均明显减少,而粒细胞[(22.4±7.8)％比(71.5±20.3)％,P＜0.05]、髓系来源抑制细胞[(5.4±2.5)％比(57.5±18.8)％,P＜0.05]以及M2型肿瘤相关巨噬细胞[(1.5±1.2)％比(5.6±3.7)％,P ＜0.05]明显增多.肝转移组织伴大量炎细胞浸润(34.7±11.6)％.浸润细胞以粒细胞(47.7±12.2)％、髓系来源抑制细胞(37.4±10.3)％以及M2型肿瘤相关巨噬细胞(15.2±7.4)％为主,树突细胞(17.6±8.2)％次之,T淋巴细胞(10.6±5.5)％以及B淋巴细胞(7.8±2.9)％较少.转移灶中细胞因子呈明显Th2类型.结论 胰腺癌肝转移可引起强烈的全身和肿瘤局部免疫抑制效应,其中髓系来源抑制细胞以及M2型肿瘤相关巨噬细胞可能起到关键作用.免疫环境的重建可作为胰腺癌肝转移防治的重要靶点.