目的 检测乙酰水杨酸(acetylsalicylic acid,ASA)对牙龈干细胞(mesenchymal stem cells derived from gingiva,GMSCs)免疫调节功能的影响,初步探索乙酰水杨酸对干细胞治疗免疫相关疾病疗效的提高作用及机制。方法 通过流式细胞仪分析ASA对GMSCs干细胞表面标志分子CD146、CD105、CD90、CD34 和 CD45的影响,通过BrdU掺入法以及MTT细胞实验检测GMSCs增殖率。建立 GMSCs与T细胞体外共培养系统,通过流式细胞仪分析细胞凋亡并用酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测相关炎症因子。进一步建立硫酸葡聚糖(dextran sulfate sodium,DSS)诱导性小鼠肠炎模型,通过追踪小鼠体重等临床表现以及结肠病理组织切片,研究GMSCs注射对DSS诱导肠炎的治疗作用,以研究ASA对GMSCs免疫调节功能的促进作用,并进一步探索其分子机制。结果 ASA促进GMSCs增殖并调高CD146及CD105等干细胞表面标记分子在GMSCs的表达。GMSCs与T细胞共培养中,GMSCs诱导T细胞凋亡,ASA可以增强GMSCs诱导T细胞凋亡,同时抑制T细胞分泌炎症因子干扰素γ和肿瘤坏死因子α。GMSCs注射对DSS诱导性小鼠肠炎具有治疗作用,表现为小鼠体重下降减缓、肠炎临床指数降低、结肠组织切片HE染色显示炎性细胞浸润减少及病理指数降低,ASA可以促进GMSCs注射治疗小鼠肠炎疗效。分子机制上,ASA通过调高Fas/FasL信号通路FasL的表达促进GMSCs诱导T细胞凋亡。结论 ASA增强GMSCs免疫调节功能,促进GMSCs对小鼠诱导性肠炎的治疗作用。
Objective: To analyze the role of acetylsalicylic acid (ASA) in immunomodulation of me-senchymal stem cells derived from gingiva (GMSCs), and to explore the role of ASA in enhancing the immumomodulation of GMSCs and the capacity of GMSCs to treat immune disorders and the underlying mechanism. Methods: Flow cytometry analysis were used to analyze the role of ASA in the expression of stem cells surface markers CD146, CD105, CD90, CD34 and CD45 in GMSCs,and the GMSCs proliferation was analyzed by 5-bromo-2-deoxyuridine (BrdU) staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The GMSCs and T cells co-culture system was established to analyze the role of ASA in immunomodulation of GMSCs by measuring T cell apoptosis using flow cytometry analysis and inflammatory cytokines using enzyme linked immunosorbent assay (ELISA). Further more, the dextran sulfate sodium (DSS) induced colitis mouse model was established and the mouse body weight, disease activity score, histological index and pathological change of colons were analyzed after GMSC infusion. Results: The proliferation of GMSCs and the expressions of CD105, CD146 in GMSCs were increased after ASA treatment. In the GMSCs and T cells co-culture system, GMSCs induced T cells apoptosis and inhibited interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) secretion by T cells, which were enhanced by ASA treatment. In vivo, GMSCs infusion could ameliorate DSS-induced colitis, including inhibited DSS-induced mouse body weight loss, decreased disease activity score and histological index, and decreased inflammation cells infiltration in colons, as shown by hematoxylin-eosin (HE) staining. Moreover, the therapeutic effects of GMSC infusion on DSS-induced colitis could be enhanced by ASA treatment. Mechanically, ASA treatment increased FasL expression of Fas/FasL death pathway in GMSCs to induce T cells apoptosis. Conclusion: ASA enhanced immunomodulation of GMSCs and increased the capacity of GMSCs to ameliorate DSS-induced colitis in mice.
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