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目的:对比分析脾脏、腹腔和骨髓源性巨噬细胞(SPMs、PCMs和BMs)在安静及极化状态下的差异。创新点:首次对比分析脾脏、腹腔和骨髓源性巨噬细胞在安静(M0)及极化状态(M1和M2)下的特征。方法:通过小鼠脾脏研磨及单细胞贴壁获得脾源性巨噬细胞;腹腔灌洗及细胞贴壁获得腹腔源性巨噬细胞;骨髓贴壁细胞在巨噬细胞集落刺激因子体外刺激下培养7天获得骨髓源性巨噬细胞。三种细胞即为M0型巨噬细胞,M0在干扰素及脂多糖刺激下获得M1型巨噬细胞,M0在白介素4(IL-4)刺激下获得M2型巨噬细胞。通过流式细胞仪分析三种类型巨噬细胞在三种状态下的Ⅱ类主要组织相容性复合体(MHC Ⅱ)和CD86表达差异。通过实时荧光定量聚合酶链式反应(q PCR)检测肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β)、甘露糖受体(MR)和类几丁质酶3样分子(Ym1)的表达变化。结论:骨髓贴壁细胞培养能获得最大量的同源巨噬细胞(图1和2),但表型偏向于M2型巨噬细胞(图3和4)。三种巨噬细胞均能极化为M1和M2型巨噬细胞(图5),其中SPMs具有更强的M1型极化能力,而M2型极化能力之间未见明显差异(图6)。综上所述,三种细胞无论在安静及极化状态下均具有不同的特征,骨髓可以获得大量同源性巨噬细胞,但性质不同于组织源性的脾脏和腹腔巨噬细胞。
OBJECTIVE: To compare and analyze the differences of splenic, peritoneal and bone marrow-derived macrophages (SPMs, PCMs and BMs) in resting and polarized state. Innovative point: The first comparative analysis of spleen, peritoneal and bone marrow-derived macrophages in the quiet (M0) and polarization (M1 and M2) under the characteristics. Methods: The spleen-derived macrophages were obtained by mouse spleen grinding and single-cell attachment. Peritoneal lavage and cell adhering to obtain peritoneal macrophages. Adherent cells of bone marrow were cultured in vitro stimulated by macrophage colony-stimulating factor 7 days to obtain bone marrow-derived macrophages. The three types of cells were M0 macrophages. M0 macrophages were obtained under the stimulation of interferon and lipopolysaccharide, M0 macrophages were stimulated by interleukin-4 (IL-4). The expression of MHC Ⅱ and CD86 in three types of macrophages were analyzed by flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), mannose receptor (MR) and chitinase-like 3-like molecules were detected by real-time fluorescence quantitative polymerase chain reaction ) Expression changes. CONCLUSION: Maximal amounts of homologous macrophages were obtained from bone marrow adherent cell culture (Figures 1 and 2), but the phenotype was biased toward M2 macrophages (Figures 3 and 4). All three types of macrophages were polarized to M1 and M2 macrophages (Figure 5), with SPMs having more M1-type polarization than M2-type polarization (Figure 6) . In summary, all three cells have different characteristics in both quiet and polarized state. The bone marrow can obtain a large number of homologous macrophages, but the nature is different from that of tissue-derived spleen and peritoneal macrophages.