天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (4): 343-349.doi: 10.11958/20212366

• 细胞与分子生物学 • 上一篇    下一篇

骨髓间充质干细胞来源外泌体对炎症微环境中巨噬细胞 表型及软骨细胞的调控作用

邢逸 1，窦一鸣 2，王敏 1，徐海楠 1，杨强 2，孙逊 2，赵艳红 1△   

  1. 目的 研究骨髓间充质干细胞来源外泌体（BMSCs-Exo）在炎症微环境中对巨噬细胞表型极化及软骨修复 的调控作用。方法 收集BMSCs培养上清液，差速离心法提取BMSCs-Exo，透射电镜观察外泌体形态，Western blot 检测ALG2相互作用蛋白X（Alix）、肿瘤易感基因101蛋白（TSG101）表达；使用不同剂量的BMSCs-Exo（10 mg/L和 50 mg/L）处理M1巨噬细胞（分为对照组、10 mg/L Exo组、50 mg/L Exo组）及白细胞介素（IL）-1β刺激的软骨细胞（分 为对照组、模型组、IL-1β+10 mg/L Exo组、IL-1β+50 mg/L Exo组），CCK-8测定BMSCs-Exo促巨噬细胞、软骨细胞增 殖能力；Western blot、酶联免疫吸附试验（ELISA）及实时荧光定量聚合酶链式反应（qRT-PCR）验证BMSCs-Exo对M1 型巨噬细胞表型极化的影响，炎性因子IL-1β、IL-6的表达水平，以及软骨细胞成软骨相关因子Ⅱ型胶原（COLⅡ）、 基质金属蛋白酶（MMP）-13、转化生长因子（TGF）-β1的表达水平。结果 BMSCs-Exo为圆形双层囊泡，表达Alix、 TSG101，可促进巨噬细胞增殖。与对照组比较，M1型巨噬细胞经BMSCs-Exo处理后，M1型极化标志物一氧化氮合 酶（iNOS）的表达水平降低，M2型极化标志物精氨酸酶-1（Arg-1）的表达水平升高（P＜0.05）；炎性因子IL-1β、IL-6 的mRNA及蛋白表达水平显著降低（P＜0.05）；与模型组相比，炎症软骨细胞中的COLⅡ、TGF-β1表达升高，MMP-13 表达水平降低（P＜0.05）。结论 BMSCs-Exo 可调控M1型巨噬细胞向M2型巨噬细胞极化，降低炎性因子表达水 平，促进炎症微环境中的软骨修复。
  • 收稿日期:2021-10-21 修回日期:2021-12-10 出版日期:2022-04-15 发布日期:2022-04-15
  • 通讯作者: △通信作者 E-mail：leafzh@126.com E-mail:leafzhao@126.com
  • 作者简介:邢逸（1996），女，硕士在读，主要从事骨软骨组织工程方面研究。E-mail：767267217@qq.com
  • 基金资助:
    国家自然科学基金资助项目（81871782）；天津市杰出青年科学基金（18JCJQJC47900）；天津市卫生健康委员会科技人才培育项 目（KJ20052）

The regulating effect of bone marrow mesenchymal stem cell-derived exosomes on macrophage phenotype and chondrocytes in the inflammatory microenvironment

XING Yi1, DOU Yiming2, WANG Min1, XU Hainan1, YANG Qiang2, SUN Xun2, ZHAO Yanhong1△   

  1. Objective To investigate the role of bone marrow mesenchymal stem cells-derived exosomes (BMSCsExo) in the regulation of macrophage phenotypic polarization and cartilage repair in the inflammatory microenvironment. Methods BMSCs cell culture supernatants were collected. BMSCs-Exo were extracted by differential centrifugation, and the morphology of exosomes was observed by transmission electron microscopy. Western blot assay was used to detect the expression of apoptosis-linked gene 2-interacting protein X (ALIX) and tumor susceptibility gene 101 (TSG101). M1 macrophages (grouped as the control group, the 10 mg/L Exo group and the 50 mg/L Exo group) and interleukin (IL)-1β- stimulated chondrocytes (grouped as the control group, the model group, the IL-1β+10 mg/L Exo group and the IL-1β+ 50 mg/L Exo group) were treated with different concentrations of BMSCs-Exo (10 mg/L and 50 mg/L). The pro-proliferative capacity of BMSCs-Exo was determined by CCK-8. Western blot assay, enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to verify the effect of BMSCs-Exo on the phenotypic polarization of M1 macrophages, expression levels of inflammatory factor IL-1β, IL-6, and chondrogenesis-related factor collagen (COL)Ⅱ, matrix metalloproteinase (MMP)-13, transforming growth factor (TGF)-β1. Results After detection, the BMSCs-Exo were round double-layered vesicles with the expression of Alix and TSG101, promoting macrophage proliferation. Compared with the control group, the expression of M1 polarization marker inducible nitric oxide synthase (iNOS) was decreased and M2 polarization marker arginase-1 (Arg-1) was increased in M1 macrophages treated with BMSCs-Exo (P＜0.05). The mRNA and protein expression of inflammatory factor IL-1β and IL-6 were significantly ameliorated (P＜0.05). Compared with the model group, COL Ⅱ and TGF-β1 expression levels were elevated and MMP-13 expression level was down-regulated in inflammatory chondrocytes (P＜0.05). Conclusion BMSCs-Exo can induce the polarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, alleviate the expression level of inflammatory factor and promote cartilage repair in the inflammatory microenvironment.
  • Received:2021-10-21 Revised:2021-12-10 Published:2022-04-15 Online:2022-04-15
  • Contact: △通信作者 E-mail：leafzh@126.com E-mail:leafzhao@126.com

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摘要： 目的 研究骨髓间充质干细胞来源外泌体（BMSCs-Exo）在炎症微环境中对巨噬细胞表型极化及软骨修复的调控作用。方法 差速离心法提取BMSCs-Exo，透射电镜观察外泌体形态，Western blot检测Alix、TSG101表达；使用不同浓度的BMSCs-Exo（10 μg/mL 和50 μg/mL）处理M1巨噬细胞及IL-1β刺激的软骨细胞，CCK-8测定BMSCs-Exo促巨噬细胞增殖能力，Western blot和qRT-PCR验证BMSCs-Exo对M1型巨噬细胞表型极化的影响，炎症相关基因IL-1β、IL-6的表达水平，以及成软骨相关因子COL II、MMP-13、TGF-β1的表达水平。结果 BMSCs-Exo为圆形双层囊泡，表达Alix、TSG101，可促进巨噬细胞增殖。与对照组比较，M1型巨噬细胞经BMSCs-Exo处理后，M1型极化标记物（iNOS）的表达水平降低，M2型极化标记物（Arg-1）的表达水平升高（P<0.05）；炎症相关基因IL-1β、IL-6的表达水平显著降低（P<0.05），炎症软骨细胞中的COL II、TGF-β1表达升高，MMP-13表达水平降低（P<0.05）。结论 BMSCs-Exo可调控M1型巨噬细胞向M2型巨噬细胞极化，降低炎症因子表达水平，促进炎症微环境中软骨修复。

关键词: 间充质基质细胞, 外泌体, 巨噬细胞, 表型, 炎症, 骨髓间充质干细胞, 软骨修复

Abstract: Objective To investigate the role of bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exo) in the regulation of macrophage phenotypic polarization and cartilage repair in the inflammatory microenvironment. Methods BMSCs-Exo were extracted by differential centrifugation, and the morphology of exosomes was observed by transmission electron microscopy, and the expression of Alix and TSG101 was detected by Western blot. M1 Macrophages and IL-1β-stimulated chondrocytes were treated with different concentrations of BMSCs-Exo (10 μg/mL and 50 μg/mL), and the pro-proliferative capacity of BMSCs-Exo was determined by CCK-8. Western blot and quantitative real-time polymerase chain reaction(qRT-PCR) were used to verify the effects of BMSCs-Exo on the phenotypic polarization of M1 macrophages, inflammation-related expression levels of genes IL-1β, IL-6, and chondrogenesis-related factors COL II, MMP-13, TGF-β1. Results After detection, the BMSCs-Exo were round double-layered vesicles with the expression of Alix and TSG101, promoting macrophage proliferation. Compared with the control group, the expression of M1 polarization marker (iNOS) was decreased and that of M2 polarization marker (Arg-1) was increased in M1 macrophages treated with BMSCs-Exo (P<0.05). The expression of inflammatory genes IL-1β and IL-6 were significantly ameliorated (P<0.05). COL II and TGF-β1 expression was elevated and MMP-13 expression level was down-regulated in inflammatory chondrocytes (P<0.05). Conclusion BMSCs-Exo can induce the polarization of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, alleviate the level of inflammatory factor expression, and promote cartilage repair in the inflammatory microenvironment and the remission of osteoarthritis.

Key words: mesenchymal stromal cells, exosomes, macrophages, phenotype, inflammation, bone marrow mesenchymal stem cells, cartilage repair

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