人脐带间充质干细胞培养上清对M1型巨噬细胞的影响及作用机制

Effect and mechanism of human umbilical cord mesenchymal stem cell culture supernatant on M1-type macrophages

目的通过研究人脐带间充质干细胞(hucMSCs)培养上清对M1型巨噬细胞的影响,探讨其作用机制。方法选择小鼠单核巨噬细胞白血病细胞系(RAW264.7)巨噬细胞,将其种植于6孔板中,待汇合度约70%时进行处理。配制含50%hucMSCs培养上清的高糖DMEM条件培养基(CM)。巨噬细胞分成3组,blank组用2 ml高糖DMEM培养基培养24 h后更换2 ml高糖DMEM培养基培养24 h,DMEM组用2 ml含200 ng/ml脂多糖(LPS)的高糖DMEM培养基刺激24 h后更换2 ml高糖DMEM培养基培养24 h,CM组用2 ml含200 ng/ml LPS的高糖DMEM培养基刺激24 h后更换2 ml CM培养24 h。收集各组培养上清和巨噬细胞进行后续实验。利用PKH67染色剂进行CM中含细胞膜的成分染色(绿),DAPI染色剂进行巨噬细胞胞核染色(蓝),通过荧光显微镜观察巨噬细胞吞噬hucMSCs-CM中含细胞膜成分的情况。应用实时荧光定量PCR(RT-qPCR)和流式细胞多因子分析技术分析促炎因子TNF-α和抗炎因子IL-10表达水平的改变。应用流式细胞术鉴定经处理后巨噬细胞表型。结果在hucMSCs上清中,荧光显微镜下观察到RAW264.7吞噬CM中膜性成分且数量随时间增加而增加。在巨噬细胞极化实验中,RT-qPCR结果提示CM组抗炎因子IL-10 mRNA相对表达量低于DMEM组,且促炎因子TNF-αmRNA相对表达量低于blank组和DMEM组(P均<0.05)。流式细胞术多因子分析结果中,CM组抗炎因子IL-10水平低于DMEM组,促炎因子TNF-α水平低于blank组和DMEM组(P均<0.05)。流式细胞术鉴定结果中,CM处理的RAW264.7中的F4/80+CD206+CD86-巨噬细胞即M2型巨噬细胞比例高于DMEM组。结论巨噬细胞可能通过吞噬hucMSCs培养上清中膜性成分诱导M1型巨噬细胞向M2型巨噬细胞极化,从而促进抗炎反应。

Objective To evaluate the effect and investigate the mechanism of human umbilical cord mesenchymal stem cells(hucMSCs) culture supernatant on M1-type macrophages. Methods The mouse macrophage-like cell line RAW264.7 was used as the macrophage cell line, planted in the 6-well plate and treated when the confluence reached approximately 70%. Conditional DMEM medium(CM) containing 50% hucMSCs culture supernatant was prepared. Macrophages were divided into three groups. In the blank group, the cells were cultured in 2 ml DMEM medium for 24 h and then cultured in 2 ml fresh DMEM medium for 24 h. In the DMEM group, the cells were cultured in 2 ml DMEM medium containing 200 ng/ml LPS for 24 h, followed by 24-h culture in 2 ml fresh DMEM medium. In the CM group, the cells were cultured in 2 ml DMEM medium containing 200 ng/ml LPS for 24 h and subsequently cultured in 2 ml fresh CM medium for 24 h. The culture medium and macrophages from each group were gathered for subsequent experiments. Substances having cell membrane components in CM were subject to PKH67 staining(green) and RAW264 with DAPI staining(blue). The phagocytosis of cell membrane components in hucMSCs-CM by macrophages was observed under fluorescence microscope. The expression levels of pro-inflammatory factor TNF-α and anti-inflammatory factor IL-10 were analyzed by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and flow cytometry multi-factor analysis. The phenotype of macrophages was identified by flow cytometry. Results Fluorescence microscopy revealed that RAW264.7 phagocytosis substances containing cell membrane components in the CM group were increased with time in the hucMSCs supernanant. In macrophage polarization experiment, RT-qPCR indicated that the expression level of anti-inflammatory factor interleukin-10(IL-10) mRNA in the CM group was significantly lower than that in the DMEM group, and the expression level of tumor necrosis factor-α (TNF-α) mRNA, a proinflammatory mediator, was significantly lower compared with those in the blank and DMEM groups(all P < 0.05). Flow cytometry multi-factor analysis showed that the expression level of anti-inflammatory factor IL-10 in the CM group was remarkably lower than that in the DMEM group, and the expression level of pro-inflammatory factor TNF-α mRNA was significantly lower than those in the blank and DMEM groups(all P < 0.05). Flow cytometry analysis showed that the proportion of F4/80+CD206+CD86-type M2 macrophages in the CM group was higher compared with that in the DMEM group. Conclusion Macrophages may induce the polarization of M1-type macrophages into M2-type macrophages by phagocytosis of the membranous components in the hucMSCs culture supernanant, thereby promoting the antiinflammatory response.