IL-38通过上调CXCL1及IL-8募集中性粒细胞促进转移病灶的形成研究

IL-38 promotes metastatic lesion formation via up-regulating CXCL1 and IL-8 induced recruitment of neutrophils

目的:探讨IL-38在肿瘤患者肿瘤组织中的表达水平及其对肿瘤转移病灶形成的影响。方法:本研究共纳入2009年1月~2014年12月我院收治的32例转移性肿瘤患者和62例原位癌患者作为研究对象。另选取43例同期在我院体检中心进行体检的健康人员作为对照。采用ELISA法(酶联免疫吸附法)检测各组研究对象血清IL-38水平。免疫组化分析转移性肿瘤患者及原位癌患者组织IL-38、CXCL1及IL-8的表达。建立小鼠肺转移模型,分别用B16F1与B16F0细胞尾静脉注射C57小鼠,然后小鼠肝脏转染IL-38,间隔48 h重复转染。两周后统计分析小鼠肺组织转移病灶数。免疫组化检测小鼠肺组织中性粒细胞浸润、CXCL1及IL-8水平。Realtime-PCR检测肺组织中趋化因子CXCL1与IL-8的表达。肺转移模型小鼠转染IL-38后,给予小鼠CXCL1及IL-8抑制剂。比较各组小鼠肺组织转移病灶数及中性粒细胞浸润情况。Western blot检测各组小鼠肺组织中IL-38、CXCL1、IL-8的表达及MEK、ERK的磷酸化水平。结果:肺转移肿瘤患者的血清IL-38水平显著高于原位癌及健康者(P<0.05),其肿瘤组织中IL-38、CXCL1及IL-8的表达水平均显著高于原位癌患者(P<0.05)。IL-38能显著增加B16F1在C57小鼠肺部形成转移病灶的数量(P<0.05),且能促进不易发生转移的B16F0在肺部形成明显的转移病灶。组化结果显示,IL-38能显著增加小鼠肺组织中性粒细胞的浸润(P<0.05)。Realtime-PCR结果显示,IL-38转染能显著增加肺组织中CXCL1及IL-8的表达(P<0.05)。给予CXCL1及IL-8抑制剂能显著抑制IL-38引起的小鼠肺部病灶数增加及肺组织中性粒细胞浸润(P<0.05)。Western blot结果显示,IL-38转染能显著上调肺转移小鼠模型肺组织中CXCL1、IL-8的表达及MEK、ERK的磷酸化水平,给予CXCL1及IL-8抑制剂能显著抑制IL-38对MEK、ERK磷酸化的影响。结论:肺转移肿瘤患者的血清和肿瘤组织中IL-38水平均显著升高,IL-38可能通过上调CXCL1、IL-8及MEK、ERK的磷酸化水平募集中性粒细胞,从而促进转移病灶的形成。

Objective：To investigate the expression level of IL-38 in the tumor tissues and explore its effect on metastatic lesion formation. Methods：A total of 32 patients with metastatic tumor and 62 patients with carcinoma in situ in our hospital from January2009 to December 2014 were recruited as study object. Another 43 cases of healthy people who underwent physical examination in the physical examination center of our hospital were selected as control. Serum levels of IL-38 in different groups were determined using a commercially available sandwich ELISA（ Enzyme-Linked Immuno Sorbent Assay）. The expression level of IL-38,CXCL1 and IL-8 in the tissue of patients with metastatic tumor and carcinoma in situ were analysis by immunohistochemistry（ IHC）. Construction of lung metastasis mice model,B16 F1 and B16 F0 were injected into C57 mice through tail vein respectively,then the liver of mice were transfected with IL-38,and the transfection were repeated for each 48 hours. The number of metastatic lesion in the lung tissues of mice were counted and analyzed. IHC were used to evaluate the degree of neutrophil infiltration and the expression of CXCL1 and IL-8 in mice lung tissues. Realtime-PCR were used to detect the expression level of CXCL1 and IL-8 in mice lung tissues. After transfection of IL-38,CXCL1 and IL-8 inhibitors were given to the lung metastasis mice model. The number of metastatic lesion and neutrophil infiltration in different groups were compared. Western blot were used to detect the expression level of IL-38,CXCL1,IL-8 and the phospholation level of MEK,ERK. Results：The serum level of IL-38 in patients with lung metastatic tumor were significant higher than in patients with carcinoma in situ and healthy control（ P〈0. 05）. The expression level of IL-38,CXCL1 and IL-8 in the tissue of patients with metastatic tumor were significant higher than in patients with carcinoma in situ（ P〈0. 05）. IL-38 transfection significant increased the number of metastatic lesions formed in the lung of B16 F1 injected C57 mice（ P〈0. 05）,and promoted the formation of metastatic lesion of B16 F0 which was difficult to metastasis. The results of histochemistry showed that IL-38 significant increased neutrophil infiltration in the lung tissue（ P〈0. 05）. The results of realtime-PCR showed that IL-38 transfection significant increased the expression level of CXCL1 and IL-8（ P〈0. 05）. The increasing number of metastatic lesions and neutrophil infiltration induced by IL-38 transfection were significant abolished by using CXCL1 and IL-8 inhibitors（ P〈0. 05）. The results of Western blot showed that IL-38 transfection significant increased the expression level of CXCL1,IL-8 and the phospholation level of MEK,ERK in the lung tissue,while the effect of IL-38 to MEK and ERK were significant abolished after given CXCL1 and IL-8 inhibitors. Conclusion：The level of IL-38 of serum and tumor tissue in patients with lung metastatic tumor increased significantly. IL-38 may promotes the formation of metastatic lesions via up-regulating CXCL1 and IL-8 induced recruitment of neutrophils.