滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应

摘要

BACKGROUND: There are mesenchymal stem cells (MSCs) in synovium that have the ability to proliferate extensively in culture. OBJECTIVE: To study the method of isolating and culturing human synovium-derived mesenchymal stem cells (SMSCs), to explore the immune-phenotype and the effect of SMSCs on the proliferation of T lymphocytes, and to evaluate the immunological characteristics of SMSCs.METHODS: Cell populations were enzymatically released from the synovial membrane obtained from human knee joints by arthroscopy. Monoclone was selected as primary SMSCs. SMSCs were assayed on its multiplication capacity and cell vitality and characterized by FACS analysis. According to different ratios of SMSCs to peripheral blood monocytes, SMSCs were treated with mitomycin in the MLR system. The lymphocyte proliferation rate was measured by MTT assay and then the inhibition rate was calculated.RESULTS AND CONCLUSION: There was no statistical significance between the 10 clones in multiplication capacity and cell vitality (P > 0.05). SMSCs were positive for CD44, CD90, CD105, but negative for CD14, CD34, CD45 and HLA-DR, respectively. After the second passage, CD166 appeared to be positive. The proliferation of T lymphocytes could be inhibited to different extents by adding different ratios of SMSCs to human PBMCs, and the inhibition rate increased as the ratios of SMSCs to PBMCs increased. Human SMSCs can be isolated from knee joint. These cells have the ability to proliferate extensively in culture, express the phenotype of MSCs and exhibit immune regulatory effects.%背景:滑膜组织中可分离出具有多向分化潜能干细胞特性的细胞.目的:探索滑膜间充质干细胞体外分离、培养的可行性、免疫表型的鉴定及对混合淋巴细胞反应体系中淋巴细胞增殖的影响,评价其免疫学特性.方法:关节镜下获取10例半月板损伤患者的膝关节滑膜组织,胶原酶消化获得有核细胞.挑选单细胞克隆,筛选获得滑膜间充质干细胞.检测其增殖能力、细胞活力,流式检测其细胞免疫表型,采用人双向混合淋巴细胞反应体系及植物血凝素刺激的淋巴细胞增殖反应体系,根据滑膜间充质干细胞与外周血单个核细胞的不同比例加入丝裂霉素处理滑膜间充质干细胞.MTT法检测并计算其抑制率.结果与结论:10组滑膜间充质干细胞系增殖能力和细胞活力差异无显著性意义(P > 0.05).10组细胞系免疫表型:CD44、CD90、CD105呈阳性,CD14、CD34、CD45和HLA-DR呈阴性.滑膜间充质干细胞抑制混合淋巴细胞反应体系中及植物血凝素刺激的淋巴细胞增殖反应体系中淋巴细胞的增殖、抑制率与加入的滑膜间充质干细胞数量成正比.提示关节滑膜组织可以分离、培养获得滑膜间充质干细胞,其具有间充质干细胞特异性表型,且滑膜间充质干细胞具有免疫调节作用.