神经生长因子对神经干细胞分化及神经元轴突形成的影响_乌优图

主要观察指标：通过巢蛋白、神经元特异烯醇化酶、胶质原纤维酸性蛋白免疫组化染色对培养细胞进行鉴定。检测神经元
数量及轴突数量、长度。
结果：所培养出的细胞团均为巢蛋白阳性，诱导分化后均可产生神经元特异烯醇化酶、胶质原纤维酸性蛋白阳性细胞。诱
导分化第 6 天，诱导组神经元数量、单个神经元轴突数量、最长轴突长度均明显高于对照组（t=3.301，2.982，4.012，
Wu YT, Wang YJ.Effects of nerve growth factor on the differentiation of neural stem cells and the formation of neuronal axons.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):5631-5635 [www.zglckf.com/zglckf/ejournal/upfiles/08-29/29k-5631(ps).pdf]
P
均< 0.01）。
结论：神经生长因子可促进神经干细胞向神经元的分化，还可以增加由神经干细胞分化而来的神经元突起数量及长度。
关键词：神经干细胞；神经生长因子；神经元；轴突
乌 优 图 ， 王 运 杰 . 神 经 生 长 因 子 对 神 经 干 细 胞 分 化 及 神 经 元 轴 突 形 成 的 影 响 [J]. 中 国 组 织 工 程 研 究 与 临 床 康 复 ， 2008 ，
110001, Liaoning
Province, China
Wu You-tu ★ ,
Studying for master’s
degree, Department of
Neurosurgery, First
Affiliated Hospital,
China
Medical
University, Shenyang
分为 2 组滴入培养板，对照组加入 10%FBS，诱导组加入 10%FBS+50 μg/L 神经生长因子，培养 5~7 d。连续观察 5 个神
经元特异烯醇化酶染色阳性且未与其他神经元发生连接的孤立神经元，求助于以神经元为圆心的同心圆，分别计数内径为
37.5 μm 和 75 μm 圆环内的突触数量，将两者均值视为神经元轴突数量，通过此同心圆测量最长轴突的长度。
通讯作者：王运 杰，博士，教授， 中国医科大学附 属第一医院神经 外科，辽宁省沈阳 市 110001 wangyunjie@ yahoo.cn
中图分类号:R394.2 文献标识码:A 文章编号:1673-8225
(2008)29-05631-05
收稿日期：2008-04-11 修回日期：2008-06-20
12(29):5631-5635
[www.zglckf.com/zglckf/ejournal/upfiles/08-29/29k-5631(ps).pdf]
Department
of
Neurosurgery, First
Affiliated Hospital,
China
Medical
University, Shenyang
110001, Liaoning
Province, China
wuyoutu@sina.com
Correspondence to:
Wang Yun-jie, Doctor,
Professor, Department
of Neurosurgery, First
Affiliated Hospital,
China
基础医学
乌优图，王运杰
Effects of nerve growth factor on the differentiation of neural stem cells and the formation of neuronal axons
Wu You-tu, Wang Yun-jie
Abstract BACKGROUND: The differentiation of neural stem cells (NSCs) is an important for NSCs to be applied in clinical treatment. And whether the neurons differentiated from NSCs can be connected with other neurons or not comes into being a critical problem. OBJECTIVE: To observe the effect of nerve growth factor (NGF) on the growth and differentiation of in vitro cultured NSCs, and on the formation and growth of axons. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Equipment Center of China Medical University from June 2007 to December 2008. MATERIALS: Three 2-3 day male Sprague Dawley rats were used in this study. NGF was purchased from Peprotech. METHODS: NSCs were isolated from neonatal rats by enzyme digestion and mechanical separation. At the fourth passage, cell clone masses received nestin immunocytochemistry. Remaining cells were dispersed by mechanical separation. Monoclone NSCs were incubated by limiting dilution assay, and made into 108 L-1 monoplast suspension in complete medium. NSCs were assigned into 2 groups. NSCs in the control group were incubated in 10% fetal bovine serum (FBS). NSCs in the induction group were incubated in the 10% FBS+50 μg/L NGF for 5-7 days. The five isolated neurons with positive expression of neuron specific enolase (NSE) were observed. The number of axons was measured through concentric circles (37.5 μm and 75 μm diameter) circling neurons to detect the length of long axon. MAIN OUTCOME MEASURES: Cultured cells were identified by nestin, NSE and glial fibrillary acidic protein (GFAP) immunohistochemistry to test the number of neurons, number and length of axons. RESULTS: Neurospheres were Nestin-positive and could differentiate into the NSE-positive or GFAP-positive cells. At day 6, the numbers of neurons and axons were significantly more, and the length of longest axons was significantly longer in the induction group than in the control group (t=3.301, 2.982, 4.012, P < 0.01). CONCLUSION: NGF can induce the differentiation of NSCs into neurons, and increase the number and length of the axons of the neurons differentiated from NSCs.
0 引言
(54200804110012/ ZS·Q)
ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH
5631
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乌优图，等. 神经生长因子对神经干细胞分化及神经元轴突形成的影响
应 用 要 点 ： ①成功地在体外培养出 神经干细胞，并对传统的细胞分离方 法进行改进。②以往国内外实验证明 神经生长因子对已成熟轴突具有促进 其再生、出芽和分枝的作用，本实验 初步证明神经生长因子对神经干细胞 分化而来的初级神经元也具有促进其 轴突形成的作用，拓展了人们对神经 生长因子的认识。
偏 倚 或 不 足 ：实验初步论 证了神经生长因子可以促 进初级神经元轴突形成和 生长，应进一步研究在此过 程中神经生长因子所采用 的是何种信号传导途径，究 竟引发了什么样的作用机 制来促进神经元初期突起 的形成和生长。
重 要 的 概 念 ：初级神经元是指那些新 生的、未极化的或未完全成熟的神经元。 轴突的出芽分枝是指由神经元极化后直 接形成的一级轴突再次出现分枝的现象。 神经元的极化现象是指初级神经元由开 始的圆形或椭圆形状态向不均衡、不规则 形状演变的过程。生长锥是初级神经元向 不规则形状发生极化时伸出的锥形突起， 生长锥不断延长最终形成轴突。
Medical
University来自百度文库 Shenyang
110001, Liaoning
Province, China
wangyunjie@yahoo.cn
Received:2008-04-11 Accepted:2008-06-20
中国医科大学附 属第一医院神经 外科，辽宁省沈阳 市 110001
乌优图★，男， 1981 年生，内蒙 古自治区赤峰市 人，蒙古族，中国 医科大学在读硕 士，主要从事神经 再生方面的研究。 wuyoutu@ sina.com
材料：清洁级二三天龄新生 SD 雄性大鼠 3 只，神经生长因子为 peprotech 公司产品。
方法：酶消化和机械分离法相结合体外分离培养新生鼠神经干细胞，传至第 4 代的细胞克隆团行巢蛋白免疫细胞化学染色
观察，剩余细胞团用机械法分散，采用有限稀释法进行单克隆神经干细胞培养，加入完全培养基制成 108 L-1 的单细胞悬液，
摘要
背景：将大量的神经干细胞定向诱导分化后的神经细胞能否与其他神经细胞建立功能联系是目前解决神经干细胞应用于临
床的重要问题之一。
目的：观察神经生长因子对体外培养的神经干细胞生长和分化的影响，以及神经生长因子对神经轴突形成和生长的作用。
设计、时间及地点：细胞学体外观察，于 2007-06/2008-12 在中国医科大学设备处完成。
中国组织工程研究与临床康复 第 12 卷 第 29 期 2008–07–15 出版 Journal of Clinical Rehabilitative Tissue Engineering Research July 15, 2008 Vol.12, No.29
神经生长因子对神经干细胞分化及神经元轴突形成的影响★