Objective It is to establish a simple and efficient method for the culture and identification of brain derived neural stem cells in neonatal SD rats. Methods ①Cerebral cortex (isolated from the neonatal SD rats,in 2 - 3 days)was cut into small pieces (about 1 mm3 )with elbow operation knife,then filtered through a 200 mesh nylon net to form a single cell suspension,suspension cultured in serum - free medium including growth factor,and passaged by the method combined Try-pLTM Express digestion with mechanical blowing. ②On the 5th passage cells,the specific surface markers Nestin of NSC and its differentiated cells (stimulated by 5% fetal bovine serum)were identified by immunofluorescence staining. Results The re-sults of culture and identification of neural stem cells:neonatal SD rats (2 - 3 days)brain-derived cells could be proliferated and formed spherical aggregates in vitro,and can be passaged continuously and steadily. The neurosphere cells at fifth genera-tion were postive for Nestin and were capable of differentiating into NSE-positive cells and GFAP-positive cells (stimulated by 5% fetal bovine serum). Conclusion The method can be used to cultivate and identify the neural stem cells from neonatal SD rat brain easily and efficiently.%目的 建立一种简便、高效的新生大鼠脑源性神经干细胞的培养与鉴定方法.方法 ①用弯头手术刀将分离出的新生SD大鼠(2~3 d)脑组织切成小碎块(约1 mm3),经200目尼龙网过滤形成的单细胞悬液在含有生长因子的无血清专用培养基内悬浮培养,并且使用TrypLTM Express消化联合机械吹打法进行传代.②对传至第5代的培养细胞,通过免疫荧光染色法鉴定神经干细胞的特异性表面标志物Nestin及经5%胎牛血清诱导分化后NSE、GFAP的表达.结果 新生SD大鼠(2~3 d)脑源性细胞在体外能够大量增殖并形成球状聚集物,并且能够连续、稳定的传代.传至第5代的细胞Nestin免疫荧光染色阳性,5%胎牛血清诱导分化后出现NSE、GFAP免疫荧光染色阳性的细胞.结论 该方法可简便高效的培养和鉴定新生大鼠脑源性神经干细胞.
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