硫酸右旋糖苷通过抑制M2型巨噬细胞极化以减弱M2型巨噬细胞对HGC-27胃癌细胞侵袭和迁移的促进作用

Dextran sulfate attenuates the promoting effect of M2-type macrophages on the invasion and migration of HGC-27 gastric cancer cells by inhibiting the polarization of M2-type macrophages

目的:探讨硫酸右旋糖苷(dextran sulfate,DS)对M2型巨噬细胞极化以及M2型巨噬细胞促进胃癌细胞侵袭和迁移的影响,观察M2型巨噬细胞在胃癌组织中的浸润情况并评估其临床意义。方法:采用佛波酯(phorbol 12-myristate 13-acetate,PMA)、白细胞介素4(interleukin-4,IL-4)和IL-13体外诱导人单核细胞THP-1极化为巨噬细胞(M0和M2型),并应用免疫细胞荧光及蛋白质印迹法进行验证。在巨噬细胞诱导和极化过程中加入DS,采用蛋白质印迹法检测DS对M2型巨噬细胞标志物CD206和CD163蛋白表达的影响,Transwell小室迁移实验检测DS对巨噬细胞趋化募集能力的影响。建立巨噬细胞与HGC-27胃癌细胞共培养模型,将HGC-27胃癌细胞与经DS处理或未处理的巨噬细胞(M0和M2型)进行共培养,然后分别采用划痕愈合实验和Transwell小室侵袭实验检测HGC-27胃癌细胞的迁移和侵袭能力,应用蛋白质印迹法检测侵袭和迁移相关蛋白的表达情况。采用免疫组织化学法检测人胃癌组织中M2型巨噬细胞的浸润情况,并分析浸润程度与临床病理特征之间的关系。结果:采用PMA、IL-4和IL-13成功诱导THP-1细胞极化为M0和M2型巨噬细胞。DS可以抑制M2型巨噬细胞的极化过程,下调M2型巨噬细胞中CD206和CD163蛋白的表达水平(P<0.001和P<0.05),以及抑制M2型巨噬细胞的趋化募集能力(P<0.001)。M2型巨噬细胞可以促进HGC-27胃癌细胞的迁移和侵袭能力,并且上调HGC-27胃癌细胞中N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)的表达水平(P<0.05或P<0.001)。DS干预M2型巨噬细胞的极化过程后,可以明显减弱M2型巨噬细胞对HGC-27胃癌细胞侵袭和迁移的促进作用(P<0.01或P<0.001)。人胃癌组织中M2型巨噬细胞的浸润程度高于正常胃黏膜组织(P<0.01),并且M2型巨噬细胞的浸润程度与胃癌的分化程度和分期相关(P均<0.05)。结论:DS能够抑制M2型巨噬细胞的极化,减弱其对胃癌细胞趋化募集的应答能力,抑制M2型巨噬细胞对胃癌细胞侵袭和迁移能力以及侵袭和迁移相关蛋白表达的促进能力。

Objective:To investigate the effects of dextran sulfate(DS)on the polarization of M2-type macrophages and the promotion of M2-type macrophages on the invasion and migration of gastric cancer cells,and to observe the infiltration of M2-type macrophages in gastric cancer tissues and to evaluate its clinical significance.Methods:Phorbol 12-myristate 13-acetate(PMA),interleukin-4(IL-4)and IL-13 were used to induce the polarization of human monocyte THP-1 into macrophages(M0-type and M2-type)in vitro,and the polarization results were verified by immunocytofluorescence and Western blotting.DS was added during macrophage induction and polarization,and the effects of DS on the expressions of M2-type macrophage markers CD206 and CD163 proteins were detected by Western blotting.The effects of DS on the chemotactic recruitment of macrophages were detected by Transwell migration assay.The co-culture model of macrophages and HGC-27cells was established by Transwell method.The macrophages(M0-type and M2-type)treated with or without DS were co-cultured with gastric cancer HGC-27 cells,and the migration and invasion of HGC-27 cells were detected by Wound healing assay and Transwell invasion assay,respectively.The expressions of invasion and migration-related proteins were detected by Western blotting.Immunohistochemical method was used to detect the infiltration of M2-type macrophages in human gastric cancer tissues,and the relationship between the infiltration and the clinicopathological characteristics was evaluated.Results:The THP-1 cells were successfully polarized into M0-type and M2-type macrophages by PMA,IL-4 and IL-13.After DS interfered with the polarization of M2-type macrophages,the expression levels of CD206 and CD163 in M2-type macrophages were decreased(P<0.001,P<0.05),and the chemotactic recruitment ability of M2-type macrophages was significantly reduced(P<0.001).After co-cultured with M2-type macrophages,the migration and invasion abilities of HGC-27 cells and the expressions of N-cadherin,vimentin and matrix metalloproteinase-2(MMP-2)in HGC-27 cells were significantly increased(P<0.05,P<0.001).After the polarization of M2-type macrophages was interfered by DS,the promotion effects of M2-type macrophages on the invasion and migration of HGC-27gastric cancer cells were inhibited(P<0.01,P<0.001).The infiltration degree of M2-type macrophage in human gastric cancer tissues was higher than that in normal gastric tissue(P<0.01),and the infiltration degree of M2-type macrophage was associated with the differentiation degree and clinical stage of gastric cancer(P<0.05).Conclusion:DS can inhibit the polarization of M2-type macrophages,weaken their response to chemotactic recruitment of gastric cancer cells,and inhibit the ability of M2-type macrophages to promote the invasion and migration of gastric cancer cells and the expression of invasion and migration-related proteins.