IL-6在巨噬细胞向M2型极化过程中的诱导作用

目的 将小鼠巨噬细胞系RAW264.7细胞和骨髓瘤细胞系KM3细胞共培养,探讨IL-6在巨噬细胞向M2型极化的过程的作用.方法 取对数生长期细胞,分为3组:A组(KM3细胞组),B组(RAW264.7细胞组),C组(RAW264.7细胞+KM3细胞组).分别予以ACTA(IL-6特异性抑制剂激活素A)或rIL-6(重组人IL-6)处理后,检测肿瘤相关M2型巨噬细胞表达标志F4/80+ CD206+的比例.RT-PCR和Westernblot方法检测各组细胞因子CCL22、IL-10、IL-12、TNF-αmRNA和蛋白表达量.ELISA法检测各组细胞培养上清中IL-6的含量.结果 RAW264.7和KM3细胞共培养24 h后,与对照组相比,M2 型巨噬细胞比例显著增加(P<0.05);予以ACTA处理后M2型巨噬细胞表达明显下降(P<0.05);而予以rIL-6处理后M2型巨噬细胞表达明显上调(P<0.05).与B组(RAW264.7细胞组)相比,C组(RAW264.7细胞+KM3细胞)M2型巨噬细胞相关细胞因子CCL22,IL-10的mRNA和蛋白表达水平明显上调(P<0.05),而M1型巨噬细胞相关因子IL-12,TNF-α的mRNA和蛋白表达水平明显下调(P<0.05).与A组、B组相比,C组细胞上清液中IL-6含量在24 h、48 h、72 h时间点均明显上调(P<0.05).通过浓度梯度改变RAW264.7细胞或KM3细胞的数量,发现RAW264.7细胞数量的改变显著影响了IL-6的表达水平.结论 将RAW264.7细胞和KM3细胞共培养后,后者能诱导RAW264.7细胞向肿瘤相关M2型巨噬细胞转化, IL-6在上述转化过程中发挥重要作用.

The induction effect of IL-6 in polarization process of macrophages to M2 type in vitro

Objective To investigate the effects of IL-6 in polarization process of macrophages to M2 type,with macrophage cell line-RAW264.7 of mice being co-cultured with myeloma cell line KM3 in vitro.Methods The cells at exponential growth phase were divided into three groups: group A (KM3 cell),group B (RAW264.7 cell),group C (RAW264.7 cell +KM3 cell).After the cells were treated with ACTA (a specific inhibitor of IL-6) or rIL-6 (recombinant human IL-6),respectively,the proportion of F4/80+ CD206+ cells was detected.The expression levels of CCL22, IL-10, IL-12, TNF-α lpha were measured by RT-PCR and Western Blot, respectively.The content of IL-6 in culture supernatant was detected by ELISA.Results After RAW264.7 cells and KM3 cells were co-cultured for 24 hours, as compared with that in control group, the proportion of M2 type macrophages was significantly increased ( P <0.05),moreover after the cells were treated by ACTA, the expression of M2 type macrophages was obviously decreased ( P <0.05),however, after the cells were treated by rIL-6, the proportion of M2 macrophages was significantly increased ( P <0.05).As compared with those in group B, the expression levels of M2 macrophage related cytokines-CCL22 and IL-10 mRNA and protein were significantly up-regulated in group C ( P <0.05),but the expression levels of M1 macrophage related cytokines-IL-12, TNF-α lpha mRNA and protein were obviously down-regulated ( P <0.05).As compared with those in group A and group B, the levels of IL-6 in culture supernatant were significantly increased at 24h, 48h, 72h time points in group C ( P <0.05).When the cell counts of RAW264.7 cells or KM3 cells were changed by different concentration gradient, the expression levels of IL-6 were more easily influenced by the chages of RAW264.7 cell number.Conclusion After RAW264.7 cells are co-cultured with KM3 cells in vitro, the latter can induce the transformation of RAW264.7 cell into tumor-associated M2 macrophages, moreover, IL-6 may play an important role in the transformation process.