IL-6在巨噬细胞向M2型极化过程中的诱导作用

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

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。

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 24 h,48h,72 h 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.