中文摘要：

目的探讨生理和炎性微环境下人脐带间充质干细胞（human umbilical cord derived mesenchymal stem cells,h UCMSCs）能否通过外泌体诱导调节性T细胞的生成来发挥免疫抑制功能。方法胶原酶消化法分离h UC-MSCs,应用流式细胞术鉴定h UC-MSCs。IFN-γ模拟体内炎性微环境,以未预处理为对照,分别提取外泌体,得到Nor-h UC-exo和IFN-γ-stimulated h UC-exo,分析其浓度及粒径分布等特征、并鉴定表面标记蛋白CD63的表达。采用h UC-MSCs、Nor-h UCexo、IFN-γpretreated-h UC-MSCs、IFN-γstimulated h UC-exo与人外周血单个核细胞共培养5 d后,流式细胞术检测T细胞的增殖、调节性T细胞的比例变化。结果 h UC-MSCs高表达CD73、CD44等间充质干细胞标志物。IFN-γ刺激前后外泌体粒径无明显变化,但IFN-γ刺激后分泌量和CD63增加（P〈0.05）。CFSE染料示踪结果显示,h UC-MSCs来源的外泌体抑制PBMCs增殖（P〈0.01）,且IFN-γ刺激后明显提高外泌体的免疫抑制能力（P〈0.01）。在活化T细胞中,IFN-γ-stimulated h UC-exo组Treg比例（11.53±0.88%）与IFN-γpretreated-h UC-MSCs组（7.54±0.50%）（P〈0.05）、Norh UC-exo组（6.60±0.56%）（P〈0.01）、对照组（3.87±0.73%）（P〈0.01）相比升高。结论 h UC-MSCs可通过分泌外泌体来发挥免疫调节作用,在炎症因子刺激下h UCMSCs分泌的外泌体明显促进调节性T细胞的生成,h UC-exo可能是潜在的免疫抑制载体。

英文摘要：

Aim To investigate whether human umbilical cord mesenchymal stem cells（ h UC-MSCs） exposed to inflammatory conditions could release large amounts of exosomes to induce regulatory T cells（ Treg）. Methods h UC-MSCs were isolated by enzyme digestion method.（ In vitro） interferon γ（ IFN-γ） was added into h UC-MSCs to mimic inflammatory microenvironments,then exosomes were extracted from the supernatant of normal conditional medium or IFN-γ pretreated h UC-MSCs. Both sources of exosomes,Nor-h UC-exo and IFN-γ-stimulated h UC-exo, were identified by Nanoparticle Trafficking Analysis（ NTA） and Western blot for the exosome-enriched protein CD63. Next,human peripheral blood mononuclear cells（ PBMCs）stimulated with PHA were respectively co-cultured with h UC-MSCs,IFN-γ-pretreated-h UC-MSCs,h UC-MSCs exosomes or IFN-γ-stimulated-h UC-MSCs exosomes for5 days to assess the exosomes-T cells communication.The proliferation rate of PBMCs and frequency of CD4＋/ CD25＋/ Foxp3＋Treg were measured by flow cytometry. Results The isolated cells from human umbilical cord tissue, which were positive for CD73,CD44,CD29,CD90 and HLA-ABC,but were negative for CD31 and CD34,were mesenchymal stem cells indeed. After IFN-γtreatment,h UC-MSCs secreted numerous exosomes（ P 0. 05）. Morerover,there was a significantly higher level of CD63,but no difference in diameter between Nor-h UC-exo and IFN-γ-stimulated h UC-exo. IFN-γ-stimulated h UC-exo had a superior ability compared with Nor-h UC-exo to suppress the proliferation of PHA stimulated PBMCs due to their upregulation of the percentage of Treg（ 11. 53 ± 0. 88% vs6. 60 ± 0. 56%,P 0. 01）. Conclusion h UC-MSCs could promote the expression of Treg to modulate immunosuppression through exosomes,especially for IFN-γ-licenced exosomes,which might carry much immunotherapeutic potential.