新生小鼠神经干细胞的分离、培养和鉴定

目的：探讨新生小鼠神经干细胞(NSCs)分离、培养以及鉴定的方法。方法：分离新生昆明种小鼠(出生24h内)的大脑组织，经胰酶消化加机械吹打后，利用无血清培养基悬浮培养细胞，获得具有自我增殖能力的细胞克隆，并将细胞克隆贴壁培养测其分化能力。应用抗Ncstin、Musashi、SSEA-1、NSE免疫细胞化学方法及BrdU标记方法鉴定细胞克隆。结果：从新生昆明种小鼠大脑组织分离的细胞悬液，经悬浮培养，生成大量具有增殖能力的细胞，可形成神经球(neurosphercs)，抗Nestin、Musashi、SSEA-1阳性，BrdU标记也呈阳性。细胞克隆贴壁培养后神经球分化为神经细胞，抗NSE阳性。结论：上述方法分离的细胞具有自我更新、自我复制及分化为神经细胞的能力，属于中枢神经系统干细胞。

Isolation, culture and identification of the neural stem cells in neonatal mice

Aim: To isolate, cultivate and identify the neural stem cells (NSCs) from the neonatal mice (<24 h). Methods: The brain tissues of neonatal Kunming mice were dissocia (ted) enzymatically and mechanically. Cell suspensions were cultivated with serum-free culture medium by suspension culture technique in order to acquire large numbers of self-proliferating cell clones. And cell clones are adherently cultivated to detect differentiated capability. Immunocytochemical stainings of anti-Nestin, Musashi, SSEA-1, NSE, and BrdU labeling method were used to identify the cells clones. Results: Single cell suspensions, which were dissociated from the neonatal mice and were cultivated by suspension culture, had potential of self-proliferation, and were able to form large numbers of cell groups, namely neurospheres. These clones were Nestin-, Musashi-and SSEA-1-positive and also a part of the cells were BrdU-positve. Neurospheres were able to differentiate nerve cells after adherent culture. Conclusion: The nerve cells isolated and cultivated in our study have potential of self- renewal, self-proliferation, and differntiation. And they are the stem cells of central nervous system (CNS).