不同无血清培养体系对人脐带间充质干细胞生物学功能的影响

Effects of different serum-free culture systems on the biological function of hUC-MSCs

目的 探讨3种不同无血清培养体系对人脐带间充质干细胞(hUC-MSC)体外培养的生物学功能的影响。方法 取3株脐带组织,各分3组实验组,经贴壁法分别置于3组不同无血清培养体系(A组:纯化学成分合成的无血清培养体系,B组:血小板裂解物+基础培养基的无血清培养体系,C组:纤维蛋白原包被+含重组人蛋白质的无血清培养体系)中培养至第3代,比较其细胞增殖能力、细胞表型、三系分化能力以及免疫调节功能。结果 3组实验组细胞均呈长而扁平的梭形,贴壁生长,大小均一。其增殖曲线趋势相似,A组和B组P2、P3代次的增殖能力优于C组(P均<0.05)。3组实验组细胞均符合国际干细胞协会对hUC-MSC细胞表面标志物鉴定的要求,A组和B组P3代次的CD29和CD90的表达量均高于C组(P均<0.05),并且3组实验组均具有三系分化能力。3组实验组中P3代次的T淋巴细胞增殖率、Th1和Th17细胞亚群分泌的细胞因子比例均低于对照组,调节性T细胞(CD4+CD25+FOXP3+)比例均高于对照组,而3组实验组之间两两比较差异无统计学意义(P均> 0.05)。结论 3种不同无血清培养体系的hUC-MSC均能保持其基本的生物学特性和免疫调节活性,纯化学成分合成的无血清培养体系和血小板裂解物+基础培养基的无血清培养体系的增殖能力和细胞表面标志物表达均优于纤维蛋白原包被+含重组人蛋白质的无血清培养体系。

Objective To evaluate the effects of three different serum-free culture systems on the biological function of human umbilical cord mesenchymal stem cells(hUC-MSC)cultured in vitro.Methods Three strains of umbilical cord tissues were obtained,divided into 3 experimental groups and cultured to the 3rd generation(P3)in three different serum-free culture systems(Group A:serum-free culture system synthesized by pure chemical components,group B:serum-free culture system consisting of platelet lysate+basic culture medium,group C:serum-free culture system consisting of fibrinogen coating+recombinant human protein).The cell proliferation,cell phenotype,three-line differentiation and immunomodulatory function were compared.Results All the cells in three groups were long andflat fusiform,adherent to the wall and uniform in size.The proliferation curve trend was similar,and the proliferation ability of P2 and P3 generations in groups A and B was better than that in group C(both P<0.05).The cells in three experimental groups all met the requirements of the International Stem Cell Society for identification of hUC-MSC cell surface markers.The expression levels of CD29 and CD90 in P3 generations in groups A and B were higher than those in group C(all P<0.05),and all three experimental groups had the ability of three-line differentiation.Compared with the control group,the proliferation rate of T lymphocytes in P3 generation and the proportion of cytokines secreted by Th1 and Th17 cell subsets were significantly decreased in the three experimental groups,and the proportion of regulatory T cells(CD4+CD25+FOXP3+)was significantly increased in the three experimental groups,but there was no statistical significance among three experimental groups(all P>0.05).Conclusions The hUC-MSC in three groups can maintain basic biological characteristics and immunomodulatory activity.The proliferation ability and expression levels of cell surface markers in groups A and B are superior to those in group C.