口腔医学研究

口腔医学研究 ›› 2019, Vol. 35 ›› Issue (5): 497-501.DOI: 10.13701/j.cnki.kqyxyj.2019.05.020

• 口腔干细胞与口腔生物学研究 • 上一篇    下一篇

骨桥蛋白对巨噬细胞极化影响的体外实验研究

冯丽华1, 王敏1, 夏海斌1*, 黄开耀2   

  1. 1. 武汉大学口腔医院种植科 湖北 武汉 430079;
    2. 中国科学院水生生物研究所 湖北 武汉 430070
  • 收稿日期:2018-09-25 出版日期:2019-05-28 发布日期:2019-05-21
  • 通讯作者: 夏海斌,E-mail:xhaibin@whu.edu.cn
  • 作者简介:冯丽华(1992~),女,湖北恩施人,硕士在读,主要从事口腔基础及临床的研究工作。
  • 基金资助:
    国家自然科学基金(编号:31570982)

Effect of Osteopontin on Polarization of Macrophage in Vitro.

FENG Li-hua1, WANG Min1, XIA Hai-bin1*, HUANG Kai-yao2   

  1. 1. Department of Oral Implantology,Hospital of Stomatology,Wuhan University,Wuhan 430079,China;
    2. Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430070,China.
  • Received:2018-09-25 Online:2019-05-28 Published:2019-05-21

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摘要： 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 factor α, 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。

关键词: 骨桥蛋白, 巨噬细胞, M1型极化, M2型极化

Abstract: 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.

Key words: Osteopontin, Macrophage, M1 polarization, M2 polarization