胚胎大鼠大脑额叶皮质神经干细胞的分离、培养与鉴定-东南大学学报医学版

胚胎大鼠大脑额叶皮质神经干细胞的分离、培养与鉴定

单位：1.东南大学基础医学院,人体解剖学与组织胚胎学系,江苏,南京,210009； 2.淮安市第二人民医院,神经内科,江苏,淮安,223002； 3.东南大学临床医学院,江苏,南京,210009

分类号：R-332, Q813.11, Q254

出版年·卷·期（页码）：2006·25·第四期（248-251）

摘要：

目的:探讨从胚胎大鼠大脑额叶皮质分离培养神经干细胞(neural stem cell, NSCs)的方法及NSCS的特性.方法:将14.5d胎龄的大鼠额叶皮质脑组织制备成单细胞悬液,于含EGF 和bFGF 的DMEM/F12无血清培养液中培养,形成原代细胞球后,进行单细胞克隆传代扩增. 免疫荧光组化方法检测克隆球Nestin、Map-2、 GFAP抗体标记情况.结果:原代培养2周后,可形成大量悬浮生长的神经球,BrdU和Nestin检测呈阳性,诱导分化后呈Map-2、GFAP阳性. 结论:可从胚鼠脑皮质中分离培养纯化、扩增传代获得NSCs,经诱导后可以分化为神经元和星形胶质细胞,可作为细胞替代治疗中枢神经系统疾病的供体来源.

Objective To investigate the method of isolation,culturing,purification,expanding and identification of neural stem cells from the cerebral cortex of frontal lobe of fetal SD rat and to observe the characteristics of growth and induction of neural stem cells.Methods The cells derived from the cerebral cortex of frontal lobe in 14.5 d rat embryos were made into monocyte suspension and cultured in serum-free DEME/F12 medium containing EGF and bFGF.The NSCs of monocyte suspension were isolated and passaged,and were expanded numerously in consecutive generations.When they formed neurospheres,NSCs were identified using Nestin antibody with immunofluorescence techniques.NSCs were induced to differentiate in serum.Identification was carried out using Map-2 and GFAP antibody for differentiating NSCs—neuron from astrocytes respectively by immunoflorescence techniques.Results A lot of suspending neurosphere were observed two weeks after primary culture.By the way of monocyte cloning and expansion consecutive generations,a large quantity of purified NSCs were obtained.Nestin positive cells could be found by immunohistochemistry techniques,and its differerentiated progeny expressed Map-2 and GFAP respectively.Conclusion Purified and proliferatied NSCs could be obtained in a large quantity from the cerebral cortex of frontal lobe in fetal SD rat and the NSCs could be induced to differentiate into neurons and astrocytes in serum.

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