乳鼠海马神经干细胞的体外分离、扩增和分化研究

The Research of Isolation,Proliferation and Differentiation of Neural Stem Cells from the Newborn Rat Hippocampus in vitro

探讨海马神经干细胞(neural stem cells,NSCs)在体外分离扩增和诱导分化的可行性。无菌条件下分离新生(24 h)SD大鼠海马神经干细胞,采用无血清培养和胎牛血清诱导分化。免疫荧光染色技术分别检测诱导前细胞巢蛋白(Nestin)的表达,以及分化细胞的神经元特异性烯醇化酶(neuron specific enolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,以鉴定细胞类型。流式细胞仪检测神经干细胞分化前后增殖能力的变化。结果显示:从乳鼠海马分离培养的细胞生长状态良好,具有克隆增殖能力,并呈Nestin表达阳性,分化后可出现NSE及GFAP表达阳性的细胞。流式细胞仪检测显示:诱导前,细胞增殖活跃,S+G2/M期细胞为(36.27±1.99)%,而分化各阶段(3,7,10 d)S+G2/M期细胞比例与诱导前(Ctrl)相比则明显下降(P<0.05),分别为(26.39±1.10)%、(26.33±1.33)%和(24.54±1.12)%。这些结果表明乳鼠海马存在神经干细胞,并具有自我更新和多向分化的潜能,可用于基础和临床的相关研究。

The experiment aimed to explore the feasibility of the isolation, proliferation and differentia tion of neural stem cells （NSCs） from the hippocampus in vitro. NSCs were isolated sterilely from the hippocampus of newborn （24 h） SD rat, which were cultured in the serumfree medium and induced to differentiate by the fetal bovine serum induction medium. Immunofluorescence was performed to detect Nestin, NSE and GFAP in order to identify the cell types, respectively. And flow cytometry was used to determine proliferative activity of NSCs before and after induction. The results demonstrated that the cells isolated from the hippocampus had the ability of prolif eration and could futher form clone spheres and express Nestin. In addition, the differentiated cells expressed NSE and GFAP, respectively. Meanwhile, the results from flow cytometry showed that NSCs were actively dividing and the percentage of cells in S＋G2/M was （36.27±1.99）%. However, at development stages of differentiation （3, 7, 10 d）, the percentages of cells in S＋GJM were obviously decreased （P〈0.05）, which were （26.39±1.10）%, （26.33±1.33）%, （24.54±1.12）%, respectively. In conclusion, neural stem cells exist in the hippocampus of newborn （24 h） rat, which have the ability of selfrenewal and multipotent differentiation, and would be very useful for basic and clinic re search.