牙龈间充质干细胞衍生外泌体USP13抑制NF-κB信号通路诱导巨噬细胞M2型极化

doi:10.13701/j.cnki.kqyxyj.2026.05.009

摘要/Abstract

摘要： 目的:探讨牙龈间充质干细胞衍生外泌体(gingival mesenchymal stem cell,GMSCs-derived exosomes,Exos)中泛素特异性蛋白酶13(ubiquitin-specific protease 13,USP13)对巨噬细胞极化的调控作用。方法:分离并鉴定GMSCs-Exos,通过透射电镜观察其结构,Western blot检测Exos标记CD9和CD63表达水平。THP-1细胞经佛波酯诱导分化为巨噬细胞。免疫荧光染色法证实巨噬细胞可摄取GMSCs-Exos。将巨噬细胞分为对照组(巨噬细胞单独培养)、模型组[白细胞介素-4(interleukin-4,IL-4)]+IL-13诱导M2型极化)、GMSCs-Exos组(巨噬细胞与GMSCs-Exos共培养)、GMSCs-Exos+USP13小干扰RNA(small interfering RNA,siRNA)组(巨噬细胞与GMSCs-Exos及USP13小干扰RNA共培养)和GMSCs-Exos+核因子κB(nuclear factor-κB,NF-κB)激动剂Compound-32组(巨噬细胞与GMSCs-Exos及Compound-32共培养)。采用免疫共沉淀实验检测USP13与核因子κB抑制蛋白α(inhibitorof nuclear factor kappa Balpha,IκBα)的相互作用。Western blot检测巨噬细胞内USP13、IκBα、p65、磷酸化p65(p-p65)、M2型标志物CD206和精氨酸酶-1(Arginase-1,Arg-1)蛋白表达水平。流式细胞术检测细胞表面CD206阳性比例。酶联免疫吸附试验法测定巨噬细胞上清液中转化生长因子-β(transforming growth factor-β,TGF-β)、IL-10和Arg1的水平。结果:GMSCs-Exos为典型囊泡结构,CD9和CD63高表达。巨噬细胞内吞GMSCs-Exos后,USP13与IκBα结合,降低IκBα泛素化水平。与对照组相比,模型组和GMSCs-Exos组巨噬细胞中p-p65水平降低,核内p65水平降低、细胞质p65水平升高,M2型标志物CD206和Arg1蛋白表达水平升高,细胞表面CD206阳性比例升高,TGF-β、IL-10和Arg1水平升高。而GMSCs-Exos+Compound-32组与GMSCs-Exos组相比,上述M2型极化指标变化趋势相反。结论:本研究证实巨噬细胞内吞GMSCs-Exos后,USP13与IκBα相互作用后降低IκBα泛素化水平,进而抑制巨噬细胞p65磷酸化及核转位,促进巨噬细胞M2型极化。

关键词: 牙龈干细胞, 外泌体, 泛素特异性蛋白酶13, IκBα, 核因子-κβ

Abstract: Objective: To investigate the regulatory effect of ubiquitin-specific protease 13 (USP13) in gingival mesenchymal stem cell (GMSCs)-derived exosomes (GMSCs-Exos) on macrophage polarization. Methods: GMSCs-Exos were isolated and identified. Their morphological structure was observed via transmission electron microscopy, and the expression levels of exosomal markers CD9 and CD63 were determined using Western blotting. THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining verified the uptake of GMSCs-Exos by macrophages. Macrophages were divided into five groups: control group (macrophages cultured alone), model group (macrophages with M2 polarization induced by IL-4 and IL-13), GMSCs-Exos group (macrophages co-cultured with GMSCs-Exos), GMSCs-Exos + USP13 siRNA group (macrophages co-cultured with GMSCs-Exos and USP13 small interfering RNA), and GMSCs-Exos + nuclear factor kappa B (NF-κB) agonist (Compound-32) group (macrophages co-cultured with GMSCs-Exos and Compound-32). The interaction between USP13 and inhibitor of nuclear factor kappa B alpha (IκBα) was analyzed using co-immunoprecipitation (Co-IP). Western blotting was used to determine the expression levels of USP13, IκBα, p65, phosphorylated p65 (p-p65), and M2 markers (CD206 and Arg1) in macrophages. Flow cytometric analysis measured the proportion of CD206-positive cells on the cell surface. Enzyme-linked immunosorbent assay (ELISA) quantified the levels of transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and arginase-1 (Arg1) in the macrophage supernatant. Results: GMSCs-Exos exhibited a typical vesicular morphology and high expression levels of CD9 and CD63. Following internalization of GMSCs-Exos by macrophages, USP13 bound to IκBα, which reduced the ubiquitination level of IκBα. Compared with the control group, the model group and GMSCs-Exos group showed decreased levels of p-p65 and nuclear p65, along with increased cytoplasmic p65 levels. Additionally, these two groups exhibited elevated expression levels of M2 markers (CD206 and Arg1), an increased proportion of CD206-positive cells on the cell surface, and higher levels of TGF-β, IL-10, and Arg1. By contrast, compared with the GMSCs-Exos group, the GMSCs-Exos + Compound-32 group displayed opposing trends in the aforementioned M2 polarization indicators. Conclusion: Following internalization of GMSCs-Exos by macrophages, the interaction between USP13 and IκBα reduces the ubiquitination level of IκBα. This, in turn, inhibits the phosphorylation and nuclear translocation of p65 in macrophages, ultimately promoting macrophage M2 polarization.

Key words: gingival stem cells, exosomes, USP13, IκBα, NF-κB

高佳明, 岳佳胜, 张耐新. 牙龈间充质干细胞衍生外泌体USP13抑制NF-κB信号通路诱导巨噬细胞M2型极化[J]. 口腔医学研究, 2026, 42(5): 414-420.

GAO Jiaming, YUE Jiasheng, ZHANG Naixin. Gingival Mesenchymal Stem Cell-derived Exosomes USP13 Inhibit NF-κB Signaling Pathway and Induce M2 Polarization of Macrophages[J]. Journal of Oral Science Research, 2026, 42(5): 414-420.

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