长期传代培养人脐带间充质干细胞免疫调节功能的比较

Comparison of Immunomodulatory Functions in Human Umbilical Cord Mesenchymal Stem Cells after Long-Term Expansion

该研究针对不同代次人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)进行系统性比较,探索长期传代培养对其免疫调节功能的影响及可能的机制。hUC-MSCs反复传代培养至P5、P10、P15代,对其分化潜能和表面标志物进行鉴定,β-半乳糖苷酶染色及流式细胞术检测其衰老与凋亡状况。通过不同条件下与人外周血淋巴细胞共培养,比较不同代次hUC-MSCs对淋巴细胞的增殖活性、细胞周期变化及T细胞亚群的调节作用;ELISA检测共培养体系中TNF-α、IFN-γ、IL-1β、IL-6含量。Western blot检测不同代次hUC-MSCs中PD-L1、IDO的表达。结果表明,传代培养至P15代,其干细胞生物学特性无明显改变,但β-半乳糖苷酶染色阳性细胞数显著升高。三个代次hUC-MSCs均能调控活化后的淋巴细胞周期,并有效抑制其增殖及Th1/Th17细胞亚群,影响炎症因子的表达,且三个代次间无显著性差异。相对而言,P15代细胞表现出对Treg亚群及IL-1β的调节能力下降。长期传代培养后的hUC-MSCs仍能保持其多向分化潜能和较为满意的免疫调节活性。但不可忽视高代次细胞的衰老对Treg亚群及炎症的调节作用减弱,临床应用中宜采用P10代以内hUC-MSCs以保证治疗的安全性与有效性。

The purpose of this study was to systematically compare different passages of human umbilical cord mesenchymal stem cells,explore the effect of long-term subculture on their immune regulatory function and possible mechanisms.The cells were cultured to 5th,10th and 15th passage,and their differentiation potential and surface markers were determined.Cell senescence and apoptosis were detected byβ-galactosidase staining and flow cytometry.Co-cultured with each passages of umbilical cord mesenchymal stem cells under different conditions to compare lymphocytes proliferation,cell cycle changes and T cell subsets in human peripheral blood lymphocytes.ELISA was used to detect the concentration of TNF-α,IFN-γ,IL-βand IL-6 in the co-culture system.Western blot was used to detect the expression of PD-L1 and IDO in hUC-MSCs at different passages.The study indicated that hUC-MSCs were cultured to the 15th passage with no significant change in biological characteristics of stem cell,but the number ofβ-galactosidase staining positive cells was significantly increased.Three passages of hUC-MSCs could regulate cell cycle in activated lymphocytes,effectively suppressed proliferation and Th1/Th17 cell subsets,affected the expression of inflammatory factors.The 15th passage cells showed deregulation of Treg subsets and IL-1β.hUC-MSCs could maintain their multi-directional differentiation potential and satisfactory immunomodulatory activity after long-term subculture;however,it could not be ignored that the disability on regulation of Tregs subsets and anti-inflammatory in high-passages cells which were aging.In clinical applications,hUC-MSCs within 10th passage should be used to ensure the safety and efficacy in treatment.