巨噬细胞极化对心肌成纤维细胞活化的影响

Effects of macrophage polarization on the activation of cardiac fibroblast

目的观察巨噬细胞极化上清对心肌成纤维细胞活化的影响。方法提取SD大鼠的骨髓细胞和心肌成纤维细胞。利用巨噬细胞集落刺激因子(M-CSF)处理骨髓细胞后,加入刺激因子:M0(无刺激因子)、M1(100μg/L脂多糖+10μg/L干扰素-γ)、M2(20μg/L白细胞介素-4)诱导巨噬细胞极化。将极化后的不同型别巨噬细胞及其培养上清分别与心肌成纤维细胞共培养,分别设空白对照组、M0组、M1组和M2组,通过细胞免疫荧光检测心肌成纤维细胞中纤维化蛋白的表达水平;实时荧光定量逆转录聚合酶链反应检测巨噬细胞和成纤维细胞特征分子的表达;Western blot检测纤维化相关蛋白及转化生长因子β受体(TGFβR)、血小板衍生生长因子受体(PDGFRs)信号通路活化情况。结果经M-CSF及相应刺激因子诱导,成功获得M1和M2型巨噬细胞。细胞共培养结果显示,与M0组相比,M1组上清培养的心肌成纤维细胞中胶原蛋白1(Col1a1)和Col3a1的mRNA水平以及平滑肌肌动蛋白(α-SMA)表达水平显著降低(P<0.05),而M2组上清培养的心肌成纤维细胞中Col1a1和Col3a1的mRNA水平以及α-SMA、结缔组织生长因子(CCN2)表达水平显著升高(P<0.05)。M1组上清培养的心肌成纤维细胞中PDGFRβ蛋白磷酸化水平显著低于M0组(P<0.01),而M2组上清培养的心肌成纤维细胞中PDGFRβ蛋白磷酸化水平显著高于M0组(P<0.05)。结论M1型巨噬细胞上清能够抑制心肌成纤维细胞活化,而M2型巨噬细胞上清能够激活心肌成纤维细胞。M1型巨噬细胞抑制纤维化的作用可能与抑制PDGFRβ通路的活化有关。

Objective To observe the effects of macrophage polarization supernatant on the activation of cardiac fibroblasts.Methods Bone marrow cells and cardiac fibroblasts of SD rats were extracted.Bone marrow cells were induced to M1 and M2 by treating with macrophage colony-stimulating factor(M-CSF),and cells were divided into MO group(no stimulating factor),M1 group(100 μg/L LPS+10 μg/L INF-γ) and M2 group(20 μg/L IL-4).Different macrophages were cocultured with cardiac fibroblasts,and different macrophage supernatants were collected to culture with cardiac fibroblasts.Immunofluorescence was performed to examine the fibrotic protein expression in cardiac fibroblasts.The mRNA levels of macrophage-specific molecules,fibrosis-related genes and signaling pathways were tested by real-time PCR.The fibrosisrelated proteins and the activation of TGFβR and PDGFRs signal pathways were detected by Western blot assay.Results After treatment with M-CSF and stimulating factors,M1 macrophages and M2 macrophages were induced.Compared with the MO group,the mRNA levels of Collal and Col3 al and the protein level of α-SMA were significantly decreased in the cardiac fibroblasts treated by the supernatant of M1 macrophage group(P <0.05),while the mRNA levels of Collal and Col3 a1 and the protein levels of CCN2 and α-SMA were significantly increased in the cardiac fibroblasts treated by the supernatant of M2 macrophage group(P <0.05).The phosphorylated protein level of PDGFRβ was significantly lower in the cardiac fibroblasts treated by the supernatant of M1 macrophage group than those in the cardiac fibroblasts treated by the supernatant of M0 macrophage group(P <0.01),while phosphorylated PDGFRβ protein levels were significantly higher in the cardiac fibroblasts treated by the supernatant of M2 macrophage group than those in the cardiac fibroblasts treated by the supernatant of M0 macrophage group(P <0.05).Conclusion M1 macrophage supernatant can inhibit the activation of cardiac fibroblasts,while M2 macrophage supernatant can activate cardiac fibroblasts.The inhibitory effect of M1 macrophages on fibrosis may be related to restraining the activation of PDGFRβ pathway.