目的 探讨人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells, UC-MSCs)对巨噬细胞表型的影响及其机制.方法 提取小鼠原代巨噬细胞,分为对照组、脂多糖(lipopolysaccharide, LPS)和干扰素γ (interferon-γ, IFN-γ)诱导组、干细胞共培养组.诱导组加入LPS和IFN-γ诱导24h,干细胞共培养组在LPS和IFN-γ诱导24 h后与人脐带间充质干细胞通过Transwell共培养24 h.采用qRT-PCR和流式细胞术检测巨噬细胞炎性因子的表达,免疫荧光观察巨噬细胞表型,Western blot检测巨噬细胞极化状态和极化通路关键蛋白的表达.结果 干细胞共培养后的巨噬细胞形态改变,长梭形细胞减少,细胞上清IL-lβ、TNF-α与诱导组相比分别下降79.5％、41.1％P均＜ 0.05),抑炎因子IL-4增加91.4％(P ＜ 0.05);免疫荧光显示,干细胞共培养组Ml型巨噬细胞标记物iNOS阳性细胞比例下降32.48％, M2型巨噬细胞标记物Argl阳性细胞比例增加32.25％(P均＜ 0.05); Western blot结果 显示,参与巨噬细胞极化的PI3K-AKT通路表达增强,而抑制该通路后,巨噬细胞向M2表型极化减弱.结论 人脐带来源的间充质干细胞可促进巨噬细胞向M2表型极化,抑制炎症进展,其与PI3K-AKT通路的激活有关.%Objective To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) on macrophages phenotype transition and the potential mechanisms. Methods Macrophages were achieved from C57BL/6J mice and divided into three groups: control group, LPS and IFN- γ induced group and MSCs group. After treated with lipopolysaccharide (LPS) and interferon γ (IFN-γ) for 24 hours, macrophages were co-cultured with UC-MSCs in a Transwell system (MSCs group) or not (LPS and IFN-γ induced group). The expression of inflammatory cytokines was measured by qRT-PCR and flow cytometry. Macrophages phenotype was detected by immunofluorescence. The polarization state and PI3K/AKT signaling pathway were detected by Western blot. Results After co-cultured with MSCs, macrophages manifested morphological changes with decrease of elongated cells, and levels of IL-1 β, TNF-α in macrophages media reduced by 79.5％ and 41.1％, respectively (P ＜ 0.05, respectively), while levels of IL-4 increased by 91.4％ compared with those of induced group (P ＜ 0.05). Immunofluorescence analysis revealed that proportion of iNOS positive cells decreased by 32.48％ and proportion of Argl positives cells increased by 32.25％ in MSCs group (P ＜ 0.05, respectively). Western blot analysis further showed unregulated expression of PI3K and p-AKT in MSCs group, and macrophages polarization towards M2 type was partly blocked after inhibiting PI3K/AKT signaling pathway. Conclusion UC-MSCs can polarize macrophages into M2 type and alleviate chronic inflammation, which is related to activation of PI3K-AKT signaling pathway.
展开▼
