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目的研究人胚胎海马神经干细胞体外长期培养的条件和其在自主分化条件下的分化能力和分化特点。方法从人胚胎海马分离神经干细胞。采用无血清培养法,进行体外培养、扩增,形成神经球。使神经球贴壁分化,分化培养基不含有任何细胞有丝分裂促进剂。使用5-溴脱氧尿嘧啶核苷(BrdU)标记分裂增生的细胞,观察细胞的分裂增殖情况。使用免疫细胞化学法鉴定神经干细胞及其在不加诱导剂下的自主分化能力。结果从人胚胎海马分离的神经干细胞具有增殖能力,细胞倍增时间为3.2d。BrdU检测有正在分裂、增殖的细胞。细胞贴壁分化后可以出现Nestin、GFAP、Tuj-1表达阳性的细胞。神经干细胞共培养6个月,传代14代。结论分离培养的海马神经干细胞具有自我更新和增殖能力,可以长期培养。在不加任何诱导剂的自主分化条件下可以向神经元、胶质细胞分化。少突胶质细胞的培养需要不同的培养条件。分离培养的干细胞具有神经干细胞的特征。可用于基础和临床的相关研究。
Objective To study the conditions of long-term culture of human embryonic hippocampal neural stem cells in vitro and their differentiation and differentiation in autologous differentiation. Methods Neural stem cells were isolated from human embryonic hippocampus. Using serum-free culture, in vitro culture, amplification, the formation of neurospheres. Differentiation of neurospheres adherent, differentiation medium does not contain any cell mitosis promoter. 5-Bromodeoxyuridine (BrdU) was used to label the proliferating cells, and the cell division and proliferation were observed. Immunocytochemistry was used to identify neural stem cells and their ability to autonomously differentiate without induction. Results Neural stem cells isolated from human embryonic hippocampus had proliferative capacity with a doubling time of 3.2 days. BrdU detects cells that are dividing and proliferating. Cells adherent differentiation can appear Nestin, GFAP, Tuj-1 positive cells. Neural stem cells co-cultured for 6 months, passage 14 generations. Conclusion The hippocampal neural stem cells isolated and cultured have self-renewal and proliferative ability and can be cultured for a long time. Under the conditions of independent differentiation without any inducer, it can differentiate into neurons and glial cells. Oligodendrocyte cultures require different culture conditions. Stem cells isolated and cultured have the characteristics of neural stem cells. Can be used for basic and clinical research.