低氧预处理对脐带间充质干细胞表型、增殖及神经营养因子分泌功能的影响

摘要

目的 探讨低氧预处理对脐带间充质干细胞表型、增殖、神经营养因子分泌功能的影响.方法 收集分娩过程中丢弃的脐带组织,分离培养脐带间充质干细胞,将原代培养的间充质干细胞进行传代.取传至第3代的脐带间充质干细胞,随机分为低氧组和常氧组,分别置于氧浓度为5%、21%的恒温培养箱中培养6天.取对数生长期的两组细胞,采用流式细胞术检测CD44、CD45、CD90、HLA-DR表达,细胞免疫荧光法检测CD34及层粘连蛋白、波形蛋白表达,实时荧光定量PCR法检测脑源性神经营养因子(BDNF)、血管内皮生长因子(VEGF)、肝细胞生长因子(HGF)、睫状节神经细胞营养因子(CNTF)mRNA表达.两组培养1~6天进行细胞计数,并采用CCK-8法检测细胞增殖活性.结果 两组均高表达CD44、CD90、层粘连蛋白、波形蛋白,低表达CD45、HLA-DR、CD34,证实两组细胞均具备间充质干细胞表型特性;两组上述细胞表型相关指标表达比较P均>0.05.两组培养1~6天细胞计数及细胞增殖活性均先升高后降低;低氧组培养1~6天细胞计数均高于常氧组,培养2~6天细胞增殖活性均高于常氧组(P均<0.05).低氧组BDNF、VEGF、HGF、CNTF mRNA相对表达量均高于常氧组(P均<0.05).结论 低氧预处理对脐带间充质干细胞的表型特征无明显影响,但可提高其增殖能力及神经营养因子分泌功能.%Objective To investigate the influences of hypoxic preconditioning on cell phenotype, proliferation, and secretion of neurotrophic factors of human umbilical cord mesenchymal stem cells (UCMSCs).Methods The discarded umbilical cord tissues during delivery process were collected for UCMSCs culture, and the primary cultured mesenchymal stem cells were passaged.Cells at passage 3 were randomly divided into the normoxia group and hypoxia group, which were growing in 21% and 5% O2, respectively.Cells in logarithmic growth phase were used.The expression of CD44, CD45, CD90, and HLA-DR was detected by flow cytometry;the expression of CD34, laminin, and vimentin was determined by immunocytochemistry;the mRNA expression of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and ciliary neurotrophic factor (CNTF) was determined by real-timer fluorescent quantitative PCR.After the cells were cultured for 1 to 6 days, we did the cell counting, and determined the cell proliferation by CCK-8.Results CD44, CD90, laminin, and vimentin were highly expressed, and CD45, HLA-DR, and CD34 were low expressed in both of the two groups, which showed that cells in the two groups had the characteristics of mesenchymal stem cells.There was no significant difference in expression of phenotype-related indexes between the two groups (all P>0.05).The CCK-8 results indicated that the cell counting and proliferation rate first increased and then decreased in 1-6 days′ culture in the two groups.When the cells were cultured for 1-6 days, the cell counting in the hypoxia group was higher than that in the normoxia group;when the cells were cultured for 2-6 days, the proliferation activity of the hypoxia group was higher than that of the normoxia group (all P<0.05).The mRNA expression of HGF, BDNF, VEGF, and CNTF in the hypoxia group was higher than that of the normoxia group (all P<0.05).Conclusion Hypoxic preconditioning has little effect on the phenotypic features of UCMSCs, but can significantly enhance the proliferation rate and the secretion of neurotrophic factors.