胚胎大鼠神经干细胞培养及分化鉴定的实验研究

Experiment on cultivation and identification of neural stem cells in embryonic rats

目的建立胚胎大鼠神经干细胞体外培养及分化鉴定的方法。方法分离不问孕龄(13- 18d)SD胎鼠脑室下区、中脑及海马等部位组织,在含有表皮生长因子和碱性成纤维细胞生长因子及N-2 添加剂的DMEM培养基中悬浮培养,形成神经球体后撤除生长因子,经消化传代于含胎牛血清培养基中贴壁生长。应用免疫荧光方法检测原代细胞特异性标记巢蛋白、神经元标记微管蛋白-β、胶质细胞标记胶质纤维酸性蛋白,以及少突胶质细胞标记半乳糖脑苷的表达。结果胎鼠脑组织不同部位均可分离出神经干细胞,体外培养可以向神经元和神经胶质细胞分化,孕龄13d与18d胎鼠产生神经干细胞球体的能力不同,前者优于后者。结论 (1)胚胎大鼠脑内不同部位来源的组织生成神经干细胞球体数量和质量基本相同,但是不同胎龄的鼠脑组织产生神经干细胞球体的能力有所差异。(2)神经干细胞具有向神经元和神经胶质细胞分化的潜能。

Objective To establish the protocol of cultivation and identification of neural stem cells from embryonic rat brain in vitro. Methods Cells isolated from subventricular zone （SVZ）, midbrain and hippocampus of 13 d or 18 d SD embryonic rats were suspending cultivated in DMEM media containing of EGF, bFGF and N-2 supplement, the growth factors were withdrawn after formation of neurospheres, passage cells were cultured in media containing fetal bovine serum, on an adhesive substrate （tissue culture plastic coated with polylysine）. The expression of nestin （specific marker of primary cells）, tubulin-β （marker of neurons）, glial fibrillary acidic protein （marker of glia cells） and galactoguanosine （marker of oligodendrocytes） were determined by immunfluorescence technique. Results Neural stem cells might be isolated from different parts of embryonic rat brain and differentiated into neurons and glial cells when cultured in vitro, but the ability in producing neurospheres was different between 13 d and 18 d embryonic rat brains, the former could produce more neurospheres. Conclusion （1） The quantity and quality of neurospheres generated from different parts of embryonic rat brain is almost no difference, but the abilities in producing neurospheres were different among different fetal-aged embryos. （2） Neural stem cells have the potential to differentiate into neurons and glial cells.