成年小鼠室管膜下区神经干细胞分离及培养方法改良

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。

A modified method for the isolation and culture of neural stem cells from the adult mice subventricular zone

Objective: To modify a method for isolation and culture of the adult mice neural stem cells(NSCs) and optimize the culture conditions,so as to provide an invaluable tool for studying the proliferation,differentiation and therapeutic potential of adult stem cells.Methods:A coronal cut was made 1.5 mm rostral to the optic chiasm and the subventricular zone(SVZ) of the forbrain was dissected.The collected tissue was dissociated into single cell suspension through mechanical and chemical digestion,dissociated c...