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目的 :探讨M2型巨噬细胞在肺腺癌A549细胞迁移和侵袭中的作用及其可能涉及的信号通路和作用机制。方法 :采用佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导THP-1细胞,使其分化为M2型巨噬细胞。通过Transwell小室建立M2型巨噬细胞与A549细胞的体外非接触式共培养模型,并收集M2型巨噬细胞与A549细胞共培养后的培养液。分别采用划痕愈合实验和Transwell小室侵袭实验检测M2型巨噬细胞与A549细胞共培养液处理A549细胞(CO-A549细胞)后对其迁移和侵袭能力的影响。蛋白质印迹法检测CO-A549细胞中血管内皮生长因子受体(3vascular endothelial growth factor receptor 3,VEGFR3)及血管内皮生长因子C(vascular endothelial growth factor-C,VEGF-C)蛋白表达水平的改变。采用特异性针对VEGFR3的抑制剂MAZ51处理CO-A549细胞后,再用划痕愈合实验和Transwell小室侵袭实验检测抑制VEGFR3表达后CO-A549细胞迁移和侵袭能力的改变;蛋白质印迹法检测对其细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase,ERK)活性[磷酸化ERK(phospho-ERK,p-ERK)]的影响。最后,采用RT-PCR法检测MAZ51和丝裂原活化蛋白激酶(mitogenactivated protein kinase,MAPK)/ERK信号通路抑制剂U0126作用于CO-A549细胞后对基质金属蛋白酶2 m RNA表达水平的影响。结果 :佛波酯诱导THP-1细胞分化为M2型巨噬细胞;M2型巨噬细胞与A549共培养液能促进A549细胞的迁移和侵袭能力,并上调VEGFR3和VEGF-C的表达水平(P值均<0.01);抑制VEGFR3表达后,CO-A549细胞的迁移(P<0.01)和侵袭能力(P<0.05)均被降低,p-ERK的表达水平明显下调((P<0.05);MAZ51和U0126作用于CO-A549细胞后基质金属蛋白酶2 m RNA水平均被明显降低(P值均<0.01)。结论:经过分化诱导THP-1细胞获得的M2型巨噬细胞,可通过提高VEGFR3的表达水平而促进肿瘤细胞的迁移和侵袭能力,其机制可能与活化MAPK/ERK信号通路有关。
Objective : To investigate the role of M2 macrophages in migration and invasion of lung adenocarcinoma A549 cells and the signal pathways and mechanisms involved. Methods: THP-1 cells were induced by phorbol 12-myristate 13-acetate (PMA) and differentiated into M2 macrophages. The non-contact co-culture model of M2 macrophages and A549 cells was established through the Transwell chamber, and the culture medium co-cultured with M549 macrophages and A549 cells was collected. The effect of treatment of A549 cells (CO-A549 cells) on the migration and invasion abilities of M2 macrophages and A549 cell co-cultivation liquids was examined by scratch healing test and Transwell chamber invasion assay, respectively. The expression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGF-C) protein in CO-A549 cells was detected by Western blotting. CO-A549 cells treated with MAZ51, a specific inhibitor of VEGFR3, were treated with scratch healing test and Transwell chamber invasion assay to detect the change of migration and invasion ability of CO-A549 cells after VEGFR3 expression. Western blot was used to detect the cells. Extracellular signal-regulated protein kinase (ERK) activity (phospho-ERK, p-ERK) effect. Finally, the effects of MAZ51 and mitogen-activated protein kinase (MAPK)/ERK signaling pathway inhibitor U0126 on the expression of MMP-2 mRNA in CO-A549 cells were detected by RT-PCR. RESULTS: Phorbol ester-induced THP-1 cells differentiated into M2 macrophages; M2 macrophage and A549 co-culture media promoted the migration and invasion of A549 cells and up-regulated the expression levels of VEGFR3 and VEGF-C. The average value was <0.01); after inhibiting the expression of VEGFR3, CO-A549 cell migration (P<0.01) and invasive ability (P<0.05) were all decreased, and p-ERK expression was significantly downregulated (P<0.05); MAZ51 The mRNA level of MMP-2 mRNA was significantly decreased after treatment with U0126 in CO-A549 cells (P<0.01).CONCLUSION: M2-type macrophages obtained by differentiation-induced THP-1 cells can be increased by increasing VEGFR3. The level of expression promotes the migration and invasion of tumor cells, and its mechanism may be related to the activation of MAPK/ERK signaling pathway.