吉西他滨化疗对免疫健全小鼠Panc02胰腺癌的作用以及对免疫环境的影响

The efficacy of gemcitabine on the immunocompetent murine Panc02 pancreatic cancer and its regulatory roles on the tumor immune environment

目的 建立免疫健全小鼠Panc02胰腺癌皮下种植瘤模型.利用该模型观察吉西他滨化疗对免疫健全小鼠胰腺癌的作用以及对全身及肿瘤局部免疫环境的影响.方法 利用C57BL/6J小鼠同源Panc02胰腺癌细胞建立皮下种植瘤模型.待肿瘤生长至75 ～ 100 mm3时,将荷瘤小鼠分为化疗组和对照组.化疗组利用吉西他滨50 mg/kg腹腔内注射化疗,每周2次,共4周.绘制肿瘤生长曲线,最终称量荷瘤小鼠体质量、肿瘤重量及脾脏重量.流式细胞计数检测外周血与肿瘤组织中10个免疫细胞群的变化.实时荧光定量反转录-聚合酶链反应检测荷瘤小鼠脾脏及肿瘤组织中7种细胞因子水平.免疫组织化学法及Western blot检测CD34及淋巴管内皮透明质酸受体-1（LYVE-1）蛋白表达,并计数肿瘤组织中微血管密度（MVD）及淋巴管密度（LVD）.结果 化疗组肿瘤体积明显小于对照组,在各个时间点比较差异有统计学意义（P＜0.05）.化疗组荷瘤小鼠体质量明显小于对照组[（21.00±1.88）g比（28.36±1.06）g,P＜0.01].化疗组终末肿瘤重量明显小于对照组[（641.67 ±289.92） mg比（1 492.00±462.73）mg,P＜0.01].化疗组与对照组脾脏重量比较差异无统计学意义（P＞0.05）.化疗后外周血中CD11c^＋树突状细胞增多[（22.93±2.26）％比（16.53±2.68）％,P＜0.05],CD11b^＋ Gr-1^＋髓系来源抑制细胞（MDSC）减少[（3.00±0.10）％比（7.03±0.32）％,P＜0.01].化疗后肿瘤组织中CD3^＋T淋巴细胞[（10.70±1.21）％比（21.10±3.54）％,P＜0.01]及MDSC[（5.10 ±2.11）％比（10.50±0.72）％,P＜0.05]减少,而树突状细胞[（17.13±3.21）％比（10.43±1.60）％,P＜0.05]、CD19^＋B细胞[（17.13±2.68）％比（7.90±1.87）％,P＜0.01]、Gr-1^＋粒细胞[（79.50 ±5.86）％比（46.00±3.75）％,P＜0.01]、CD3^＋NK1.1^＋自然杀伤T细胞（NKT）[（9.77±1.56）％比（4.90±1.81）％,P＜0.05]增多.化疗后脾脏中白细胞介素-4 （IL-4）表达升高（P＜0.05）,而肿瘤坏死因子-α（TNF-α,P＜0.05）及IL-2（P＜0.01）表达下降.化疗后肿瘤组织中IL-4及转化生长因子（TGF）-β表达升高（P＜0.05）,而IL-6、干扰素（IFN）-γ、TNF-α及IL-2表达均下降（P＜0.05或P＜0.01）.化疗后肿瘤组织中MDSC效应产物精氨酸酶-1（Arginase-1）明显下降（P＜0.05）.化疗后肿瘤组织中MVD（18.47±2.61比30.40±3.92,P ＜0.05）及LVD（6.66±2.77比16.27±2.02,P＜0.01）均下降.结论 吉西他滨可以抑制小鼠Panc02胰腺癌生长以及肿瘤组织中淋巴管及血管的生成,但也诱导产生了肿瘤局部和全身的免疫抑制效应,以肿瘤局部免疫抑制作用尤为明显.化疗诱导产生的免疫抑制效应以及肿瘤血管生成减少导致的肿瘤组织中血药浓度下降,可能是影响胰腺癌疗效的重要原因,有望成为提高胰腺癌化疗效果的重要靶点.

Objective To establish an immunocompetent pancreatic cancer subcutaneous bearing murine model and clarify the roles of gemcitabine on this tumor and its possible regulatory roles on the systemic and local tumor immune environment.Methods The C57BL/6J mice synergic pancreatic adenocarcinoma cell line Panc02 cell was subcutaneously implanted to establish the immunocompetent murine pancreatic cancer model.When the tumors grew to 75-100 mm^3,the tumor bearing mice were subdivided into the control group and chemotherapy group.For the chemotherapy group,gemcitabine was admitted 50 mg/kg intraperitoneally twice a week,totally for 4 weeks.The tumor growth curve was depicted and after the mice were sacrificed,the body weight,tumor weight and spleen weight were measured and compared.Flow cytometry was adopted to detect 10 immune cell populations in tumor tissue and peripheral blood.Real time quantitative reverse transcription-polymerase chain reaction was used to measure the relative mRNA level of 7 cytokines in tumor tissue and spleen.The CD34 and lymphatic vessel endothelial hyaluronan receptor 1 （LYVE-1） were labeled by immunohistochemistry and Western blotting,and then the microvessel density （MVD） and lymphatic vessel density （LVD） were analyzed.Results The tumor volume of chemotherapy group was significantly lower than that of the control group at each time point （P 〈0.05）.The body weight of chemotherapy group was significantly lower than that of the control group [（21.00 ± 1.88） g vs.（28.36 ± 1.06） g,P 〈0.01].The tumor weight of chemotherapy group was significantly lower than that of the control group [（641.67 ± 289.92） mg vs.（1 492.00 ± 462.73） mg,P 〈 0.01].The weight of spleen of the two groups was not significantly different.Compared to the control group,the CD11 c^ ＋ dendritic cell population [（22.93 ± 2.26） % vs.（16.53 ± 2.68） %,P 〈 0.05] in peripheral blood was significantly elevated,however,the CD1 1 b^ ＋ Gr-1^ ＋ MDSC population significantly declined [（3.00 ±0.10）% vs.（7.03 ±0.32）%,P 〈0.01].After chemotherapy,in tumor tissue,the CD3^ ＋ T lymphocyte population [（10.70 ± 1.21） % vs.（21.10 ± 3.54） %,P 〈 0.01] and myeloid derived suppressor cells （MDSC） [（5.10 ± 2.11） % vs.（10.50 ± 0.72） %,P 〈 0.05] declined,however,the dendritic cell population [（17.13 ± 3.21） % vs.（10.43 ± 1.60） %,P 〈 0.05],CD19^ ＋ B lymphocyte population [（17.13 ± 2.68） % vs.（7.90 ± 1.87） %,P 〈 0.01],Gr-1^ ＋ granulocyte population [（79.50 ±5.86）% vs.（46.00 ±3.75）%,P〈0.01] and CD3^＋ NK1.1^＋ natural killer T cells （NKT） population [（9.77 ± 1.56） % vs.（4.90 ± 1.81） %,P 〈 0.05] elevated.After chemotherapy,in spleen,the interleukin-4 （IL-4） expression elevated（P 〈0.05）,however,the tumor necrosis factor-α （TNF-α） （P 〈 0.05） and IL-2 expression （P 〈 0.01） declined.After chemotherapy,in tumor tissue,the IL-4 and transforming growth factor-β （TGF-β） expression elevated （P 〈 0.05）,however IL-6,IFN-γ,TNF-α and IL-2 declined （P 〈 0.01）.After chemotherapy,the product of MDSC,arginase-1,significantly declined （P 〈 0.05）.After chemotherapy,the MVD （18.47 ± 2.61 vs.30.40 ± 3.92,P 〈0.05） and LVD （6.66 ±2.77 vs.16.27 ±2.02,P〈0.01） both declined.Conclusion Gemcitabine can effectively inhibit the Panc02 pancreatic cancer and attenuate the angiogenesis and lymphangiogensis,however it can also induce strong systemic and local immunosuppressive effects.The induced immunosuppressive effects and the decline of drug concentration induced by the attenuated-angiogenesis are likely to be important factors to affect chemotherapy efficacy,and they could be promising targets for improving the chemotherapeutic effects.