【摘要】 目的:通过对体外培养人脐血来源非造血干细胞生长特性的观察和对细胞免疫表型的检测,探讨人脐血来源非造血干细胞的一般特性,为进一步研究人脐血来源非造血干细胞提供依据。方法:实验于2005-01/10在首都医科大学北京神经科学研究所和首都医科大学组织学胚胎学教研室实验室完成。脐带血由首都医科大学附属天坛医院提供,经密度梯度离心法分离新鲜脐带血,获取的单个核细胞进行培养。①倒置显微镜下观察细胞形态。②培养基中加入白血病抑制因子或用Mescencult间充质干细胞培养基进行培养,记录细胞生长状况。③流式细胞术分析脐血单个核细胞的免疫表型。结果:①人脐血非造血干细胞形态观察:较低密度接种细胞并不能形成大量贴壁细胞,而以5×106细胞密度接种则在48～72h后出现贴壁的梭形细胞,培养过程中细胞形态逐渐形成均一梭形。②生长特性:人脐血非造血干细胞接种5d后增殖速度加快,随后的几天内保持较快的增殖速度,15d后进入平台期。高糖DMEM培养基+胎牛血清和高糖DMEM培养基+胎牛血清+白血病抑制因子,两种培养体系在细胞增殖上未显示差异,白血病抑制因子并没有显著改善人脐血非造血干细胞生长增殖速度。③免疫表型:新鲜脐血经分离得到的单个核细胞中有少量CD34阳性细胞,CD90,CD166阳性细胞比例分别为(24.18±5.46)%和(36.44±6.26)%;人脐血非造血干细胞体外培养10d左右无CD34阳性细胞,CD166阳性细胞的比例达(76.48±4.54)%,较单个核细胞中CD166阳性细胞比例明显增高,并且表达HLA-ABC,而少量表达HLA-DR。结论:合适的细胞接种密度有利于人脐血非造血干细胞贴壁生长。白血病抑制因子并不能增加体外培养的人脐血非造血干细胞的生长增殖速度。人脐血非造血干细胞体外培养10d左右可以获得纯度较高的CD166阳性细胞。更多还原

【Abstract】 AIM To probe the characteristic of non-hematopoietic stem cells from human cord blood CB-nHSCs by observing its growing characteristics cultured in vitro and detecting the immune phenotype so as to provide evidence for further studies on CB-nHSCs. METHODS The experiment was conducted at Beijing Institute for Neuroscience Beijing Central Neural Regeneration and Repairing and the Department of Histology and Embryology Capital University of Medical Science between January and October 2005. Human umbilical cord blood was provided by Beijing Tiantan Hospital. The mononuclear cells MNCs divided from umbilical cord blood UCB by density gradient centrifugation were cultured. ①Morphology of CB-nHSCs was observed by inverted microscope. ②CB-nHSCs were cultured in media with leukaemia inhibitory factor LIF or Mescencult mesenchymal stem cells to record the growth condition. ③Flow cytometry was used to detect the immune phenotype of MNCs. RESULTS ①Morphology characteristics Inoculated cells with low density could not form a great amount of adhered cells which were observed cultured in 5×106 cells density after 48-72 hours and turned into even spindle shape during culture. ②Growth characteristic After 5 days inoculation the CB-nHSCSs propagated quickly and kept the speed until 15 days later. There was no difference in proliferation of CB-nHSCSs between cultured in HG-DMEM and HG-DMEM+LIF. ③Immune phenotype Flow cytometry showed that MNCs from UCB had little CD34+ cells and the percentage of positive cells for CD90 and CD166 was 24.18±5.46% and 36.44±6.26% respectively while after 10 days culture in vitro there was no CD34+ cells and the percentage of CD166+cell increased to 76.48±4.54% which was higher than that in freshly separated MNCs. At same time part of CD166+ cells was positive for HLA-ABC while few for HLA-DR. CONCLUSION Appropriate cells inoculated density can enhance adherence of CB-nHSCs and LIF has no effect on proliferation of CB-nHSCs. Comparatively pure CD166+ cells can be obtained from CB-nHSCs after 10 days culture in vitro.更多还原