胚胎大鼠神经干细胞的分离培养及自然分化的初步观察

目的神经干细胞体外培养的成功是进一步研究其分化机制的基础,从胚胎大鼠脑室下区分离、培养、鉴定神经干细胞(neuralstemcells,NSCs),并观察其向神经元的分化情况。方法分离胚胎SD大鼠脑室下区的组织,采用无血清原代及传代培养方法,获得具有克隆能力的细胞群;应用免疫细胞化学方法鉴定神经干细胞并检测分化后特异性成熟神经细胞抗原的表达;应用流式细胞检测技术观测神经干细胞随时间向神经元的分化情况。结果从胚胎大鼠脑室下区分离培养的细胞群具有克隆增殖能力,表达神经上皮干细胞蛋白(nestin),分化后的细胞表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原;随着贴壁后分化时间的延长,表达nestin的细胞数量从80.5%下降到10.9%,而神经元特异性烯醇化酶(neuronspecificenolase,NSE)阳性细胞数量则从1.1%上升到31.6%。结论用本方法分离的细胞具有自我更新和增殖能力,并具有多分化潜能,是中枢神经系统的干细胞;随着分化时间的延长,神经干细胞数量下降,而神经元比例有明显上升。

Isolation and differentiation of neural stem cells from the central nervous system of embryonic rats

Aim Culture in vitro of neural stem cells is necessary to study the mechanisms of differentia tion of neural stem cells.Here we iso late neural stem cells from the subventricular zone of embryonic rat and observe the differentiation of neural stem c ells to neuron.Methods The embryonic rat subventricular zone were dissociated mechanically and enzymatically and cell clones were acquired by using se rum-free primary and passage cultur e.Immunocytochemical staining was used to detect the specific antigen of neural stem cells and mature neural c ell after cell clones differentiate d.The differentiation rate of neural stem cells to neuron was analysed by flow c y-tometer.Results The cells isolated from subventricu lar zone of embryonic rats had the potential to form clones.These clones were nestin-positive and could differentiate into neurons,a strocytes and oligodendrocytes.Af ter ad-hesion,the number of cells which exp ress nestin decreases from 80.5%to10.9%and the number of NSE-positive cells had an conspicuous raise from1.1%to 31.6%.Conclusion The isolated cells have the ability o f self-renewal and multipotent diffe rentiation,so we can be believe that it is the stem cells of the CNS.With time of differentiation,the number of neur al stem cells go down and neurons from NSCs increase distinctively.