中文摘要：

目的研究不同分化时间对体外单层贴壁培养的大脑皮层神经干细胞（NSCs）分化成星形胶质细胞和神经元的影响。方法（1）体外分离、培养胎鼠大脑皮层原代NSCs，采用细胞免疫荧光染色检测P2代神经球和吹散成单细胞的NSCs中巢蛋白、Sox2的表达；（21取P2代神经球吹散成单细胞，接种于圆玻片上培养过夜，次日更换诱导分化培养基，分别继续培养ld、3d、5d，采用细胞免疫荧光染色检测细胞神经胶质纤维酸性蛋白（GFAPl、Tuj1的表达，分析神经元突起的平均数量、平均最长长度和平均分枝水平；（31取接种于12孔细胞培养板上的NSCs分化样品，采用实时荧光定量PCR检测NSCs不同分化时间点巢蛋白、GFAP、TujlmRNA的表达。结果（11细胞免疫荧光染色检测显示神经球、单层贴壁培养细胞几乎均为巢蛋白和Sox2双阳性细胞，说明培养的细胞几乎全部是NSCs。（21分化3d组、分化5d组NSCs中GFAP’细胞比例高于分化ld组，分化5d组NSCs中Tuil’细胞比例高于分化1d组、分化3d组，差异均有统计学意义（P〈0．05）；分化3d组、分化5d组所分化神经元突起的平均数量、平均分枝水平均高于分化1d组，差异有统计学意义（氏0．051；分化1d组、分化3d组、分化5d组所分化神经元突起的平均最长长度依次增加，差异有统计学意义（P〈0．05）；（3）与分化1d组、分化3d组比较，分化5d组NSCs中巢蛋白mRNA的表达降低，GFAP、TujlmRNA的表达增加，差异有统计学意义（P〈0．05）。结论随着分化时间的增加，体外单层贴壁培养的NSCs分化出的星形胶质细胞和神经元的数量增多，细胞形态变复杂，神经元突起也越来越成熟，巢蛋白mRNA的表达降低，GFAP和Tuj1 mRNA的表达上升。

英文摘要：

Objective To study the effects of different differentiation times （one, 3 and 5 d） on mouse embryonic neural stem cells （NSCs） differentiating into astrocytes and neurons. Methods （1） Fetal cortices of embryonic 14 d （E14） C57BL/6 mice were isolated, digested and cultured. The nestin and Sox2 expressions in the second passaged neural spheres and monolayer cultured NSCs were detected by immunofluorescent staining. （2） And then second passaged neural spheres were digested into NSCs; they were inoculated in the slide and overnight cultured, and then, they were changed into the differential medium the next day; immunofluorescence assay was used to observe the glial fibrillary acidic protein （GFAP） and Tujl expressions; the averaged numbers, mean longest length and mean branches of the neuritis were analyzed one, 3 and 5 d after differentiation. （3） Real time fluorogenic quantitative PCR was used to detect the nestin, GFA P and Tujl mRNA expressions one, 3 and 5 d after differentiation of NSCs. Results （1） NSCs were successfully cultured and almost of all cells were both nestin and Sox2 positive NSCs. （2） Immunofluorescence assay showed that as the differentiation time increasing, numbers of differentiated astrocytes and neurons became larger, and their morphologies became more complicated. The cell counting results showed that： as compared with one d group, 3 and 5 d groups had significantly high GFAP＋ astrocyte percentages （/9〈0.05）; as compared with one and 3 d groups, 5 d group had significantly higher Tuj 1＋ neuronal percentage （P〈0.05）; as compared with one d group, 3 and 5 d groups had significantly larger averaged neurite numbers （P〈0.05）; the averaged longest neurite length was increased as differentiation time increasing and there was obvious difference between each two time points （P〈0.05）; as compared with one d group, 3 and 5 d groups had significantly higher averaged neurite branching levels （P〈0.05）. （3） And PCR results mainly s