M2型TAMs激活NF-κB通路促进结肠癌细胞侵袭转移的实验研究

目的:观察M2型肿瘤相关巨噬细胞(tumor-associated macrophages，TAMs)对结肠癌LoVo细胞侵袭转移的影响，探讨其可能的作用机制。方法:建立M2型TAMs与LoVo细胞共培养体系。采用流式细胞术检测M2型TAMs生物标记分子CD163、CD206的表达情况以明确M2型TAMs诱导成功，细胞划痕实验检测M2型TAMs对细胞迁移能力的影响，酶联免疫吸附测定法(ELISA)检测细胞上清液IL-10、TGF-β1的浓度，蛋白印迹法(Western Blot）检测NF-κB通路及上皮间质转化(epithelial-mesenchymal transition，EMT)相关蛋白表达水平，免疫荧光技术检测NF-κB p65及E-cadherin入核情况。结果:流式细胞术检测结果显示，诱导后的TAMs高表达CD163、CD206；划痕实验结果显示，随着M2型TAMs浓度增加，LoVo细胞迁移能力增强(P<0.05)；酶联免疫吸附测定结果显示，随着M2型TAMs浓度增加，细胞上清液中IL-10、TGF-β1的浓度增加(P<0.05)；Western Blot结果表明，M2型TAMs可以明显上调IKKα、IKKβ蛋白表达(P<0.05)，抑制IκBα蛋白表达(P<0.05)，促进EMT相关蛋白N-cadherin、Vimentin及ZEB1的表达，抑制E-cadherin的表达(P<0.05)；免疫荧光结果显示，随着M2型TAMs浓度增加，NF-κB p65入核增多，E-cadherin入核减少。结论:M2型TAMs可以激活NF-κB通路，进而介导EMT发生。

The experimental study of M2 TAMs activating NF-κB pathway to promote the invasion and metastasis of colon cancer cells

Objective:To investigate the effect of type M2 tumor-associated macrophages（TAMs）on the invasion and metastasis of LoVo cells and its possible mechanism.Methods:Co-culture system of M2 TAMs and LoVo cells was established.Flow cytometry was used to detect the expression of biomarkers CD163 and CD206 of type M2 TAMs in order to determine the success of induction of type M2 TAMs.The LoVo cell migration ability was observed by scratch test.The content of IL-10，TGF-β1 were tested by enzyme-linked immunosorbent assay（ELISA）.Western Blot was used to detect the expression of proteins about NF-κB passway and epithelial-mesenchymal transition（EMT）.The nuclear translocation of NF-κB p65 and E-cadherin were detected by immunofluorescence.Results:Flow cytometry showed that CD163 and CD206 were highly expressed in TAMs after induction.With the increase of M2 TAMs concentration，LoVo cell migration increased(P<0.05).The concentrations of IL-10 and TGF-β1 in supernatant increased with the increase of M2 TAMs concentration(P<0.05).The results of Western Blot showed that M2 TAMs could obviously up-regulate the protein expression of IKKα，IKKβ and inhibit the protein expression of IκBα in LoVo cells(P<0.05).The protein expression of N-cadherin，Vimentin and ZEB1 can be up-regulated by M2 TAMs.The protein expression of E-cadherin can be inhibited by M2 TAMs(P<0.05).NF-κB p65 was more expressed in the nucleus with the increase of M2 TAMs，but E-cadherin was the opposite.Conclusion:M2 TAMs can promote EMT by activating NF-κB pathway.