M2亚型巨噬细胞对胃癌细胞迁移及上皮-间质转化的影响

Effect of M2 subtype macrophages on migration and epithelial-mesenchymal transition of gastric cancer cells

目的探讨M2亚型巨噬细胞对胃癌细胞迁移及上皮-间质转化(EMT)的影响。方法磁性活化细胞分选法(MACS法)分离获得临床胃癌患者肿瘤组织与外周血来源巨噬细胞。采用流式细胞和RT-qPCR分析并评价胃癌微环境中浸润的肿瘤相关巨噬细胞(TAMs)的亚型。Transwell小室迁移实验检测胃癌组织来源巨噬细胞(GC-TAMs)对肿瘤细胞迁移能力的影响。Western blot和RT-qPCR评价GC-TAMs对肿瘤细胞EMT的调节作用。结果GC-TAMs中CD204+细胞的比例明显高于癌旁组织;GC-TAMs中CD163、CD204、CD206、IL-10和CCL-22等M2亚型相关基因的转录水平明显高于癌旁组织,而M1亚型相关基因IL-23(p19)的转录水平则明显降低。胃癌患者外周血来源巨噬细胞的表面标记蛋白及亚型相关基因表达情况与GC-TAMs相似。体外共培养实验显示,M2亚型GC-TAMs对肿瘤细胞的迁移能力具有明显促进作用,镜下可见胃癌细胞形态发生明显间质细胞样改变,且细胞中EMT相关基因和蛋白fibronectin、vimentin和snail2的表达明显上调。结论胃癌微环境中浸润TAMs以M2亚型为主,且可通过诱导肿瘤细胞发生EMT而对其迁移能力发挥促进作用。

Objective To investigate the effect of M2 subtype macrophages on the migration and epithelial-mesenchymal transition(EMT)of gastric cancer cells.Methods The tumor tissue and peripheral blood derived macrophages were separated and obtained from the tumor tissue or peripheral blood in the patients with clinical gastric cancer by the magnetic activated cell sorting(MACS)method.The subtype of tumor-associated macrophages(TAMs)infiltrating in gastric cancer microenvironment was analyzed and evaluated by adopting the flow cytometry and RT-qPCR.The Transwell chamber migration experiment was used to detect the effect of gastric cancer tissue-derived tumor-associated macrophages(GC-TAMs)on tumor cell migration ability.Western blot and RT-qPCR were exploited to evaluate the regulating role of GC-TAMs on the EMT of tumor cells.Results The proportion of CD204+cells in GC-TAMs was significantly higher than that in the paracancerous tissues.The transcription levels of M2 subtype-related genes of CD163,CD204,CD206,IL-10 and CCL-22 in GC-TAMs were significantly higher than those in the paracancerous tissues,whereas the transcriptional level of M1 subtype related gene IL-23(p19)was significantly deceased.In peripheral blood macrophages from the patients with gastric cancer,the surface marker protein and the expression of subtype-related genes were similar to those in GC-TAMs.The in vitro co-culture experiment displayed that M2 subtype GC-TAMs had the significant promoting effect on the migrating ability of tumor cells.The significant interstitial-like cell morphological change of gastric cancer cells were observed under microscope,moreover the expressions of EMT-related genes such as fibronectin,vimentin and snail2 were significantly up-regulated.Conclusion Infiltrated TAMs is dominated by M2 subtype in gastric cancer microenvironment,moreover which plays the promoting role on the migrating ability by inducing the EMT occurrence of tumor cells.