大鼠胚胎脑新皮质神经干细胞的体外分离培养方法研究

Study on in vitro isolation and culture method of neural stem cells from fetal rat neocortex

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 method for isolation and culture of neural stem cells from fetal rat neocortex in vitro.Methods Neocortexes of fetal SD rats were isolated on gestational day 14.Mechanical triturations were used to improve cell dissociation status and conditions of serum-free media were optimized.Three test methods including neural stem cell marker protein（nestin and SOX2）,proliferation and clonogenic ability and the multilineage differentiation potential were employed to identify neural stem cells.Results Nestin of cells cultured with this method was expressed with strong positive,and more than 99% cells were SOX2-positive.BrdU was incorporated into the cells,and the amounts of the cells were 10.55 times as much as the initial cell number after 3 days of culture.The ratios of clonal neurosphere were（33.00 ± 4.40）% after 6 days of culture.Staining with specific protein markers in the corresponding cells confirmed that neural stem cells could form neurons（MAP2）,astrocytes（GFAP） and oligodendrocytes（O4） after differentiation induction.Conclusion Neural stem cells obtained with this method had high purity and cell output,potent self-renewal and multi-lineage differentiation ability,which will provide a good model for toxicological research.