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目的建立大鼠胚胎脑新皮质神经干细胞的体外分离培养方法。方法从孕14天SD大鼠胚胎脑分离新皮质,通过机械吹打改善细胞分散状态、优化无血清培养基条件,从而改进神经干细胞的培养方法。利用神经干细胞标志蛋白(nestin和SOX2)、增殖和克隆成球能力以及多向分化能力测试三方面对神经干细胞进行鉴定。结果以该法培养的神经干细胞nestin蛋白呈强阳性表达,SOX2阳性表达率达99%以上,BrdU掺入阳性,培养3天后细胞量为接种量的10.55倍,6日克隆成球率为(33.00±4.40)%。经相应特异性标志蛋白检测证实神经干细胞经诱导分化后可形成神经元(MAP2)、星形胶质细胞(GFAP)和少突胶质细胞(O4)。结论获得的神经干细胞纯度和细胞产出率高,具有强自我更新和多向分化能力。
Objective To establish an in vitro culture method of rat neural cortex neural stem cells in vitro. Methods The neocortex was isolated from the embryonic brain of 14-day-old SD rats and the condition of cell dispersion was improved by mechanical blowing. The conditions of serum-free medium were optimized, and the method of culturing neural stem cells was improved. Neural stem cells were identified using neural stem cell marker proteins (nestin and SOX2), proliferation and clonogenic ability and multi-directional differentiation ability test. Results The nestin protein of neural stem cells cultured in this method was strongly positive. The positive expression rate of SOX2 was over 99%. BrdU incorporation was positive. After 3 days of culture, the amount of cells was 10.55 times of that of inoculation, ± 4.40)%. The corresponding specific marker protein test confirmed that neural stem cells can form neurons (MAP2), astrocytes (GFAP) and oligodendrocytes (O4) after differentiation. Conclusion The obtained neural stem cells have high purity and high cell yield with strong self-renewal and multidirectional differentiation ability.