长期传代人脐带间充质干细胞的特性及功能

Characteristics and functions of long-term passaged human umbilical cord mesenchymal stem cells

目的:探讨传至10代(P10)的人脐带来源间充质干细胞(P10-hUC-MSC)的生物学特性及功能。方法:人脐带来源于厦门弘爱医院(伦理批号:HAXM-MEC-20201012-037-01),分离、收集、培养hUC-MSC并传代培养,收集P1-、P10-hUC-MSC,FCM检测hUC-MSC表型,细胞衰老β-半乳糖苷酶染色法及FCM法检测终末期细胞衰老与凋亡情况,秋水仙碱处理检测细胞染色体稳定性,体外成脂、成骨诱导实验检测其多向分化能力,以不同比例与外周血单个核细胞(PBMC)混合培养后FCM检测T细胞亚群及表型变化。结果:成功分离和培养的P10-hUC-MSC与P1-hUC-MSC的表型相似,表现为CD45、CD34、HLA-DR表达阴性而CD105、CD90阳性率≥95%。终末期的P1-hUC-MSC和P10-hUC-MSC均表现出β-半乳糖苷酶表达阳性和早期凋亡特征,细胞染色体核型一致且保持稳定,未发生转化现象。P1-、P10-hUC-MSC在体外都可被诱导分化成脂肪、成骨细胞。P10-hUC-MSC与PBMC以1∶1混合培养7 d后,可显著上调CD4^(+)/CD8^(+)T细胞比值、CD4^(+)Treg细胞比例和PD-1表达(均P<0.01)。结论:长期传代的P10-hUC-MSC仍然保持其生物学特性和安全性,并具备多向分化能力及免疫调节能力,这为最大限度发挥hUC-MSC的临床放疗损伤修复与预防作用提供了前期实验依据和指导。

Objective:To explore the characteristics and functions of human umbilical cord-derived mesenchymal stem cell(hUC-MSC)after 10 passages(P10-hUC-MSC).Methods:Human umbilical cord was obtained from Xiamen Hongai Hospital(ethical lot number:HAXMMEC-20201012-037-01),hUC-MSCs were isolated,collected,cultured and passaged,and P1-,P10-hUC-MSCs were collected.FCM was used to detect cell phenotypes.Senescence-associatedβ-galactosidase staining method and Annexin-Ⅴflow cytometry were adopted to detect end-stage cell senescence and apoptosis,respectively.Colchicine treatment was used to detect chromosomal stability.In vitro lipogenesis and osteogenesis induction assay was used to examine the ability of multidirectional differentiation of the cells.After co-culture with peripheral blood mononuclear cells(PBMC)at different ratios,the T-lymphocyte subsets and phenotypes were detected by FCM.Results:The phenotypes of P10-hUC-MSCs were similar to that of P1-hUC-MSCs,showing negative expression of CD45,CD34 and HLA-DR,but high positive expression rate(over 95%)of CD105 and CD90.Both groups of P1-hUC-MSCs and P10-hUC-MSCs showed positiveβ-galactosidase expression and early apoptotic characteristics,with no significant difference(P>0.05),and the cell chromosomes remained stable without transformation.Both P1-and P10-hUC-MSCs could be successfully induced and differentiated into adipocytes and osteoblasts in vitro without significant differences(P>0.05).When P10-hUC-MSCs and PBMCs were co-cultured in the ratio of 1∶1,the proportion of CD4^(+)/CD8^(+)T cells and CD4^(+)Treg cells,and PD-1 expression were all significantly upregulated(all P<0.01).Conclusion:The long-term passaged P10-hUC-MSCs still maintain their biological characteristics and safety,and possess multi-differentiation and immunomodulatory ability,which provide preliminary experimental basis and guidance for maximizing its damage repair and prevention effects in radiotherapy.