成年小鼠室管膜下区神经干细胞分离及培养方法改良  

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

作　　者：陆利[1] 朱茜[1] 夏仲年[1] 宋慧芳[1] 杨桂姣[1] 

机构地区：[1]山西医科大学人体解剖学教研室,太原030001

出　　处：《神经解剖学杂志》2011年第5期487-492,共6页Chinese Journal of Neuroanatomy

基　　金：国家自然基金(30973094);山西省青年基金(2009021045)

摘　　要：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 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 cells were then suspended in modified serum-free culture medium.NSCs were identified by nestin immunofluorescence staining.After treatment with 1% FBS（Fetal Bovine Serum）,the natural differentiation of NSCs were confirmed by immunofluorescence staining.The self-renewal and proliferation of NSCs cultured in new conditions were analyzed by serial dilution and 5-bromodeoxyuridine（BrdU） incorporation assay.Results：Using modified method,nestin positive neurospheres were obtained after 7～9 days of culture.When cultured in medium contained 1% FBS,NSCs differentiated into neurons,astrocytes and oligodendrocytes with percentages of 18.6%±3.5%,73.2%±5.2% and 3.6%±0.4%,respectively.NSCs cultured in modified conditions generated more secondary neurospheres at cell concentration of 500,1000 and 2000,compared with routine culture method（P0.05）,and BrdU-positive NSCs increased to 42.4%±6.2% in modified culture conditions（P0.05）for 8 hours BrdU labeling,indicating that the modified method facilitated self-renewal and proliferation of NSCs.Conclusion： The present stuy establishs This method provides a simple,reliable,reproducible method for isolation and culture of adult mice NSCs.

关 键 词：室管膜下区 神经干细胞 分离 培养 改良 小鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]