不同鼠龄生后大鼠海马神经干细胞的传代和分化

目的研究不同鼠龄大鼠海马神经干细胞(NSCs)的传代增殖和分化情况。方法将生后SD大鼠分为3d、10d、20d 3组,应用胰酶消化法将海马组织制成单细胞悬液,用含碱性成纤维生长因子(bFGF)、表皮生长因子(EGF)、B27的无血清细胞培养技术进行体外培养,单克隆培养后通过Nestin、NSE、GFAP免疫细胞化学染色鉴定神经干细胞、神经元、星形胶质细胞;细胞传3代后,在各组培养基中加入终浓度为10%的血清诱导分化,1周后进行NSE免疫细胞化学染色,计数阳性细胞比例,进行统计学分析。结果3组SD大鼠海马组织细胞,单克隆培养后表达Nestin,诱导分化后分别表达NSE和GFAP。生后3d大鼠神经干细胞在传1-6代时每代均快于生后10d和20d大鼠,传6代后,3组细胞传代时间间隔几近一致;生后3d组大鼠海马神经干细胞分化为神经元的比例较其它2组高(P<0.05)。结论生后3d大鼠神经干细胞增殖速度和分化为神经元的数量都高于10d和20d大鼠。

Passage and Differentiation of Neural Stem Cells from the Hippocampus of the Different Neonate Rats

Objective To investigate the proliferation and differentiation of neural stem cells(NSCs)from hippocampus of neonate rats with different ages.Methods By using trypsin digestion,neural stem cells were isolated from hippocampus of 3-day,10-day and 20-day rats.The medium of serum-free but with basic fibroblast growth factor(bFGF), epidermal growth factor(EGF)and B27 was used to culture NSCs.Immunocytochemistry for nestin was used to identify monoclone NSCs,for glial fibrillary acidic protein(GFAP)and neurone specific enolase(NSE)to identify astrocytes and neurons respectively one week after NSCs were differentiated.The percentage of the NSE positive cells was count and analyzed in statistics.Results The cultured monoclone sphere in 3 groups expressed nestin,and expressed NSE and GFAP one week after induced differentiation.NSCs of 1～6 passages isolated from 3-day rat proliferated much faster than the cells isolated from 10-day and 20-day rats,but almost same after six passage.The number of neurons differentiated from 3d rats was more than that from 10d and 20d rats(P<0.05).Conclusion NSCs from 3d rats proliferaterd faster with more number of differentiated neurons compared to 10d and 20d rats.