骨桥蛋白对巨噬细胞极化影响的体外实验研究

Effect of Osteopontin on Polarization of Macrophage in Vitro

目的:探究外源性骨桥蛋白(osteopontin,OPN)在体外对巨噬细胞极化为M1型或M2型的影响。方法:体外培养小鼠RAW264.7细胞,进行以下分组实验:(1)OPN浓度梯度实验:设定对照组,实验组加入不同浓度OPN(0.1、0.5、1.0 mg/L);(2)M1型极化实验:设定对照组、脂多糖(lipopolysaccharide, LPS)组、OPN组、LPS+OPN组;(3)M2型极化实验:设定对照组、白细胞介素-4(interleukin-4, IL-4)组、OPN组、IL-4+OPN组。培养24 h后,实时荧光定量聚合酶链反应(quantitative real-time PCR, qRT-PCR)分析M1型基因诱导性一氧化氮合酶(inducible nitric oxide synthase, iNOS)、肿瘤坏死因子-α(tumor necrosis factorα, TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)及M2型基因CD206、精氨酸酶-1(arginase-1, Arg-1)、白细胞介素-10(interleukin-10, IL-10)的mRNA表达;Western blot检测iNOS、Arg-1的蛋白表达;免疫荧光检测iNOS、CD206的表达。结果:qRT-PCR显示,与对照组比较,OPN组的M1型基因iNOS、TNF-α、IL-1β及M2型基因CD206、Arg-1、IL-10的mRNA表达均上调(P<0.05),并呈浓度依赖关系;LPS+OPN组的M1型基因表达上调最高,IL-4+OPN组的M2型基因表达上调最高(P<0.05);Western blot、免疫荧光与qRT-PCR结果一致。结论:OPN作为一种多效性细胞因子,可能促进巨噬细胞向M1型或M2型极化,并与极化诱导剂LPS和IL-4具有潜在的协同促进作用。

Objective:To study the effect of exogenous osteopontin(OPN)on the polarization of macrophage in vitro.Methods:Mouse RAW264.7 macrophages were cultured in vitro,and divided into groups as follows:(1)OPN dose-dependent experiment:the experimental group were treated with 0.1,0.5,1.0 mg/L OPN respectively in vitro;(2)M1 polarization experiment:set control group,LPS group,OPN group,LPS+OPN group;(3)M2 polarization experiment:there were control group,IL-4 group,OPN group,and IL-4+OPN group.After stimulated for 24 hours,quantitative real-time PCR(qRT-PCR)was used to measure mRNA levels of M1-type genes inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and M2-type genes CD206,Arginase-1(Arg-1),and interleukin-10(IL-10).Western blot was used to assay iNOS and Arg-1 protein.The expression of iNOS and CD206 was detected by immunofluorescence assay(IFA).Results:mRNA levels of M1 and M2-type genes both increased significantly in a dose-dependent manner on OPN group(P<0.05).The expression of M1-type genes was the highest on LPS+OPN group,while expression of M2-type genes was the highest on IL-4+OPN group(P<0.05).The Western blot and IFA results were consistent with qRT-PCR results.Conclusion:OPN may induce M1 or M2 polarization of macrophage,and has synergetic effect with LPS and IL-4,as a pleiotropic cytokine.