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目的:改良成鼠神经干细胞(NSCs)分离培养方法,优化培养条件,为系统研究成体干细胞增殖与分化特性以及利用成体干细胞进行细胞治疗奠定基础。方法:视交叉前1.5 mm处离段脑组织,分离前脑室管膜下区(SVZ),通过机械性消化与化学性消化相结合的操作方法制备细胞悬液,应用改良无血清培养基悬浮培养NSCs。nestin免疫荧光染色鉴定NSCs,1%胎牛血清诱导分化后免疫荧光染色鉴定NSCs多向分化潜能。系列稀释实验和5-溴脱氧尿核苷(BrdU)掺入实验比较改良培养与常规培养NSCs自我更新能力和增殖潜能。结果:改良培养条件,NSCs 7～9 d可形成nestin阳性神经球。应用1%FBS诱导后,NSCs分化为形态各异的细胞,包括神经元、星形胶质细胞和少突胶质细胞,分化比例分别是(18.6±3.5)%、(73.2±5.2)%和(3.6±0.4)%。系列稀释实验结果显示当细胞接种数量为500、1000和2000个,改良培养NSCs形成次代神经球数量较常规培养对照组明显增多(P<0.05),并且8 h BrdU掺入率也显著增高达(42.4±6.2)%(P<0.05),表明改良培养条件下NSCs自我更新能力和增殖活力良好。结论:本研究建立了一种简便、操作性强、重复性高的成鼠NSCs分离培养方法。
OBJECTIVE: To improve the isolation and culture methods of neural stem cells (NSCs) of adult rat and to optimize the culture conditions, which will lay the foundation for systematically studying the proliferation and differentiation of adult stem cells and the cell therapy using adult stem cells. Methods: The segmental brain tissue was isolated at 1.5 mm anterior to the optic chiasm and the SVZ was isolated. The cell suspension was prepared by mechanical digestion combined with chemical digestion. The suspension was cultured in modified serum-free medium NSCs. Nestin immunofluorescence staining to identify NSCs, 1% fetal bovine serum after differentiation induced by immunofluorescence staining to identify multi-directional differentiation potential of NSCs. Serial dilution experiments and 5-bromodeoxyuridine (BrdU) incorporation experiments compared the modified and cultured NSCs self-renewal ability and proliferation potential. Results: Under the condition of modified culture, nestin-positive neurospheres were formed in 7-9 days of NSCs. After being induced by 1% FBS, NSCs differentiated into various morphological types including neurons, astrocytes and oligodendrocytes, the differentiation rates were (18.6 ± 3.5)%, (73.2 ± 5.2)% and (3.6 ± 0.4)%. The results of serial dilution experiments showed that when the number of inoculated cells was 500, 1000 and 2000, the number of neurospheres formed by modified cultured NSCs was significantly increased (P <0.05), and the incorporation rate of 8 h BrdU was significantly higher than that of the control group 42.4 ± 6.2)%, respectively (P <0.05), indicating that the self-renewal ability and proliferative activity of NSCs under modified culture conditions are good. Conclusion: This study established a simple, operational and reproducible method of NSCs isolation and culture.