【摘要】 目的探讨神经营养素-3(NT-3)能否促进脂多糖(LPS)诱导的M1型巨噬细胞向M2型巨噬细胞极化。方法体外培养小鼠RAW264.7巨噬细胞株,分为LPS组和LPS+NT-3组。用脂多糖(100 ng/ml)刺激12h后,撤掉LPS培养基,冲洗,分别加入基础培养基和含有NT-3(40 ng/ml)的基础培养基,培养24 h后,全部置换成基础培养基培养24 h,收集上清与细胞。固定后的细胞分别做CD68/CCR7(M1型细胞标记物)、CD68/CD206(M2型细胞标记物)、CD68/Trk C免疫荧光双标染色。用酶联吸附试验(ELISA)检测上清中TNF-α和IL-10水平。结果免疫荧光细胞化学染色显示,LPS刺激后的巨噬细胞表达NT-3的受体Trk C。LPS组可观察到较多的M1型细胞和较少的M2型细胞。LPS+NT-3组可观察到M1型细胞数减少,而M2型细胞数增多。与LPS组比较,LPS+NT-3组的M1型巨噬细胞比例降低,同时M2型巨噬细胞比例增加(P<0.01)。ELISA结果显示,与LPS组比较,LPS+NT-3组上清中TNF-α的水平明显下降(P<0.01),IL-10的水平相对升高但是差异不明显(P>0.05)。结论 NT-3促进M1型巨噬细胞向M2型极化可能通过与其受体Trk C结合发挥作用,进而减少促炎因子TNF-α的分泌。更多还原

【Abstract】 Objective To investigate whether neurotrophin-3(NT-3)can promote the polarization of lipopolysaccha-ride(LPS)-induced M1 macrophages to M2 macrophages. Methods RAW264.7 macrophages were cultured in vitro and di-vided into two groups:the LPS and LPS+NT-3 groups. After stimulated with LPS(100 ng/ml)for 12 h,the medium was re-moved followed by 3 rinses with D-Hanks. Subsequently,the basal medium and NT-3 medium(40 ng/ml NT-3 in the basal me-dium)were added for 24 h.The same as above,the medium was removed and the new the basal medium was added. Then the supernatant and cells werecollected after culturing 24 h. After fixation,the cells were performed with CD68/CCR7(M1 phenotype),CD68/CD206(M2 phenotype)and CD68/Trk C double-labeled immunofluorescence-staining,respedtively. The concentration of TNF-α and IL-10 in the supernatant was detected with Enzyme-Linked Immunosorbent Assay(ELISA). Results The results of immunocyto-chemistry showed that LPS-stimulated macrophages expressed NT-3 receptor Trk C. A lot of M1 macrophages and fewer M2 macrophages were observed in the LPS group. However,in the LPS+NT-3 group,the number of M1 macrophages was de-creased and the number M2 macrophages was increased. Compared with LPS group,the ratio of M1 macrophages was signifi-cantly decreased and the ratio of M2 macrophages was increased in the LPS+NT-3 group(P<0.01). In addition,the result ofELISA showed the proinflammatory cytokines TNF-α level in LPS+NT3 group was significantly lower than LPS group(P<0.01),while the IL-10 level was only relatively increased in the LPS+NT-3 group but there was no statistical difference be-tween both groups. Conclusion NT-3 promotes the polarization of M1 macrophages to M2 phenotype mediated by binding toits receptor Trk C,and reducing the secretion of the proinflammatory cytokine TNF-α.更多还原