摘要
背景:间充质干细胞的免疫调节特性已被临床广泛应用于自身免疫性疾病和移植物抗宿主病,但是其免疫调节的可塑性导致间充质干细胞临床治疗效果出现异质性和不稳定性。目的:探索γ干扰素联合脂多糖模拟炎症微环境诱导人脐带间充质干细胞向MSC2极化的作用。方法:体外分离培养人脐带间充质干细胞,进行形态学、表面标志物、成脂及成骨诱导分化能力鉴定。分别用γ干扰素(10μg/L)、脂多糖(100μg/L)及二者联合刺激人脐带间充质干细胞24h,与植物凝集素刺激的外周血单个核细胞直接共培养或Transwell间接共培养5 d。流式细胞术检测共培养不同时间点调节性T细胞和Th1细胞比例,荧光定量PCR检测人脐带间充质干细胞的Toll样受体2,3,4 mRNA表达水平。结果与结论:①人脐带间充质干细胞呈梭形或成纤维形,高表达CD73、CD90、CD105,低表达CD34、CD45、HLA-DR;②在直接与间接共培养条件下,γ干扰素联合脂多糖刺激人脐带间充质干细胞均可增加调节性T细胞的比例,优于γ干扰素或脂多糖单独刺激组、未刺激组及外周血单个核细胞对照组(P <0.05);③Th1细胞比例随共培养时间的延长呈逐渐下降的趋势;④在间接共培养的条件下,γ干扰素联合脂多糖刺激人脐带间充质干细胞更早向免疫抑制型MSC2极化,Toll样受体3表达显著增高(P <0.05);⑤以上结果表明间接共培养体系下γ干扰素(10μg/L)联合脂多糖(100μg/L)是诱导人脐带间充质干细胞向MSC2极化的高效组合,并且MSC2的免疫抑制作用不依赖于细胞间的直接接触,为MSC2来源外泌体将来应用于临床研究奠定实验基础。
BACKGROUND: Mesenchymal stem cells have been widely applied in autoimmune diseases and graft-versus-host diseases because of their immunomodulatory capabilities. However, mesenchymal stem cells have plasticity in immunomodulation, which leads to heterogeneity and instability when used in vivo. OBJECTIVE: To investigate the polarization of human umbilical cord derived mesenchymal stem cells to an immunosuppressive phenotype(MSC2) in the inflammatory microenvironment induced by interferon-γ and lipopolysaccharide. METHODS: Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro, and then were identified by morphological characteristics, surface markers, adipogenesis and osteoinduction activity. Human umbilical cord-derived mesenchymal stem cells were treated with interferon-γ(10 μg/L), lipopolysaccharide(100 μg/L), or their combination for 24 hours, respectively, and were then co-cultured with phytohemagglutinin pre-treated peripheral blood mononuclear cells for 5 days under direct or Transwell indirect contact. The percentages of regulatory T cells and T helper 1 cells were analyzed by flow cytometry at different times. The mRNA expression levels of Toll-like receptors 2, 3 and 4 were detected by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION:(1) Human umbilical cord derived mesenchymal stem cells exhibited spindle-shaped or fibroblast-like morphology, highly expressed CD73, CD90 and CD105, and lacked expression of CD34, CD45 and HLA-DR.(2) Under direct or indirect co-culture, human umbilical cord-derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide could promote the generation of regulatory T cells, which was superior to the interferon-γ, lipopolysaccharid, un-treated and control groups(P < 0.05).(3) The percentage of T helper 1 cells gradually decreased over time.(4) Under indirect co-culture, human umbilical cord derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide were polarized into immunosupp
作者
黄恬
黄欣
赖沛龙
耿素霞
陈晓梅
王玉连
郭礼妍
曾高淳
韩凤珍
李晓红
杜欣
翁建宇
Huang Tian;Huang Xin;Lai Peilong;Geng Suxia;Chen Xiaomei;Wang Yulian;Guo Liyan;Zeng Gaochun;Han Fengzhen;Li Xiaohong;Du Xin;Weng Jianyu(School of Medicine,South China University of Technology,Guangzhou 510006,Guangdong Province,China;Department of Hematology,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,Guangdong Province,China;Department of Obstetrics,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,Guangdong Province,China;Medical Research Center of Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences,Guangzhou 510080,Guangdong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第13期2020-2027,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金面上项目(81671585,81370665)，项目负责人：翁建宇~~
关键词
人脐带间充质干细胞
Γ干扰素
脂多糖
调节性T细胞
TOLL样受体
human umbilical cord derived mesenchymal stem cells
interferon-γ
lipopolysaccharide
regulatory T cells
Toll-like receptors
