· 论著·

    [收稿日期] 2006 - 11 - 16

    [基金项目] 广东省科技联合攻关项目资助(B30502)

    [作者简介] 巨 容,女,主治医师,博士生,研究方向为危重病。

    [通讯作者] 封志纯,男,教授,博士生导师,电子信箱:zhjfengzc @126.

    com。

    人胎脑神经干细胞体外培养及分化研究

    巨 容 ,杜 江 ,兰和魁 ,王 斌 ,封志纯

    (南方医科大学附属珠江医院 儿科 ,广州 510282)

    Study on in vitro Culture and Differentiation of Neural Stem Cells from Human Embr yonic Brain

    JU Rong , DU Jiang , L A N He - kui , WA N G Bi n , FEN G Zhi - chun

    (Department of Pediat rics ,the Affiliated Zhujiang Hospital of Southern Medical University ,Guangzhou 510282 ,China)

    Abstract : Objective To study the conditions of invit ro culture ,quantities of neural stem cells(NSCs) derived f rom different location

    brain ,and abilities of differentiation of NSCs f rom human embryonic hippocampus. Methods NSCs were isolated f rom human embryonic

    hippocampus ,cortex , sub - ependymal layer ,corpus striatum. The cells were cultured and formed neuro - spheres in serum - f ree medium

    with basic fibroblast growth factor (b - FGF) ,epidermal growth factor ( EGF) ,B27 and N2 supplement . Then neuro - spheres were

    cloned and t ransferred proliferated by limited dilution. The neuro - spheres were labeled with BrdU. Differentiation happened while

    neuro - spheres adhered to dish wall . These cells were for observation of cell mitosis and proliferation. The NSCs and default differentiated

    cells were distinguished by immunocytochemisty. Results NSCs f rom human embryonic brain had ability of proliferation. Hippocampus

    was most abundant of neural stem cells ; the following was sub - ependymal layer . They could be cultured for passage and differentiate to2

    ward multiple directions. The cells labeled with BrdU had ability of mitosis and proliferation ;these cells stored in liquid nit rogen had abili2

    ty ,too. Nestin , glid fiboillary acidic protein ( GFAP) , and microtubule associated protein 2 (MAP2) expressing cells were identified after

    adhesion and differentiation via immunocytochemisty. Conclusions Embryonic brain is rich of neural stem cells which have ability of self

    - renewal and proliferation , and remained ability when they are cultured in serum - f ree medium with EGF ,b - FGF in vit ro lasting a

    long time. They are able to differentiate into neurons and ast rocytes.

    J Appl Cl in Pediatr ,2006 ,21( 24) :1709 - 1710

    Key words : embryonic ; differentiation ;neural stem cell ;human ;brain

    [摘要] 目的 研究人胚胎神经干细胞体外长期培养的条件、 不同部位脑组织神经干细胞数量及其分化能力和特点。 方法

    分离人胚胎海马、 皮质、 室周、 纹状体脑组织,培养在含碱性成纤维细胞生长因子(b2FGF) 、 表皮生长因子 ( EGF) 、 B27、 N2 添加剂

    的无血清培养基中,形成神经球,用有限稀释法进行克隆、 传代。计算神经球数量,用溴脱氧尿苷(BrdU)标记神经球,使用免疫细

    胞化学法鉴定神经干细胞自主分化能力。结果 人胚胎海马、 皮质、 室周、 纹状体部位脑组织均能培养出具有自我增殖能力的神

    经干细胞,其中海马所含神经干细胞最丰富,室周次之。BrdU 检测有正在分裂、 增殖的细胞。液氮冻存6 个月的细胞复苏后仍具

    增殖分化能力。细胞贴壁分化后可形成 Nestin、 胶质原性纤维酸性蛋白( GFAP) 、 微管相关蛋白 2 (MAP2)表达阳性细胞。 结论

    胚胎脑组织具有丰富的神经干细胞,具有自我更新和增殖能力,可长期培养。含 b2FGF、 EGF 的无血清培养基能促进神经干细胞

    连续稳定增殖, 保持神经干细胞特性。分离培养的神经干细胞可向神经元、 胶质细胞分化。

    实用儿科临床杂志,2006 ,21 (24) :1709 - 1710

    [关键词] 胚胎;分化;神经干细胞;人;脑

    [中图分类号] R729 [文献标识码] A [文章编号] 1003 - 515X(2006) 24 - 1709 - 02

    神经干细胞(NSCs)的发现和研究是目前神经科学和其他

    相关学科研究的热点之一。NSCs 是一种终身具有自我更新

    能力的细胞 ,可增殖分化出中枢神经系统(CNS)的 3 种基本细

    胞:神经元、 星形胶质细胞与少突胶质细胞。 已证实在皮层、 室

    管膜下层、 纹状体、 海马、 中脑等区域都存在神经干细胞。这

    一发现为神经系统损伤和退行性变的治疗带来了希望[ 1 ,2 ]。

    它可能替代胚胎组织而成为移植治疗 CNS疾病、 修复神经功

    能损害的新方法[ 3 ]。如何成功地进行人胚 NSCs培养并获得

    大量目的细胞 ,以供人类进行移植治疗成为关键[ 4 ]。本文系

    统地研究人胎脑 NSCs 的分离、 传代方法 ,增殖和分化特点 ......