神经干细胞的培养鉴定及分化

朱琼，女，1990年生，重庆市人，汉族，2009年第三军医大学毕业，在读硕士，主要从事神经干细胞治疗阿尔茨海默病的作用及机制研究。

通讯作者：徐亚丽，博士，副主任医师，副教授，解放军第三军医大学第二附属医院超声科，重庆市 400037

Zhu Qiong, Studying for master’s degree,

Department of Ultrasound, Second Affiliate Hospital of Third Military Medical University, Chongqing 400037, China

Corresponding author: Xu Ya-li, ., Associate chief physician, Department of Ultrasound, Second

Affiliate Hospital of Third Military Medical University, Chongqing 400037, China

神经干细胞的培养鉴定及分化

朱 琼1

，皋月娟2

，高顺记1

，陈 重1

，刘 政1

，徐亚丽1(1

解放军第三军医大学第二附属医院超声科，重庆市 400037；2

解放军第三○二医院超声科，北京市 100039)

引用本文：朱琼，皋月娟，高顺记，陈重，刘政，徐亚丽. 神经干细胞的培养鉴定及分化[J].中国组织工程研究，2017，

21(17):2708-2713.

DOI: ORCID: 0000-0002-1810-5009(朱琼) 文章快速阅读：

不仅能分化为多种类型的神经细胞替代缺失神经组织，同时能产生多种细胞因子，如脑源性神经营养因子、神经生长因子及胶质源性神经营养因子等，并促进突触发生，调节其可塑性，且能重建部分环路和功能，是神经元替代治疗的理想靶细胞。

细胞分化：指同一来源的细胞逐渐由全能到多能，最后到单能，从而产生形态功能不同的细胞群的过程，其本质是基因组在时间和空间上的选择性表达。如神经干细胞能分化为多种类型神经细胞：星形胶质细胞、小胶质细胞及神经元。同时该过程受局部微环境调节，多种理化因素都能诱导全能细胞产生分化，如血清诱导神经干细胞分化。

摘要

背景：神经干细胞在神经损伤修复和退行性疾病中有广泛的应用前景，体外培养鉴定及促神经元诱导分化是

后续研究的基础。

目的：采用悬浮神经球培养法分离培养神经干细胞并对其鉴定，了解生物学特性。 方法：采用悬浮神经球培养法从C57BL/6胎鼠大脑半球分离培养神经干细胞，观察形态特征及超微结构，CCK-8法绘制生长曲线，流式细胞仪检测细胞周期，免疫荧光法检测特异性标志蛋白Nestin 的表达；用体积分数为1%和10%的血清诱导分化后，免疫荧光法检测GFAP 、βⅢ-tubulin 和MBP 的表达。

结果与结论：①体外培养得到悬浮生长的神经球，生长曲线和细胞周期表明细胞增殖力强；②透射电镜观察到神经干细胞核浆比高，呈未分化状态；③Nestin 免疫荧光阳性；④不同体积分数的血清诱导后均可分化为星形胶质细胞、神经元和少突胶质细胞，体积分数为1%血清能诱导分化得到更多的神经元细胞；⑤结果表明，采用悬浮神经球培养法成功分离培养得到了神经干细胞，低体积分数血清有利于神经干细胞向神经元分化。 关键词：

干细胞；分化；神经干细胞；培养；鉴定；血清；诱导分化；国家自然科学基金 主题词：

神经干细胞；细胞, 培养的；血清；细胞分化；组织工程 基金资助：

Cultivation, identification and differentiation of neural stem cells

Zhu Qiong 1, Hao Yue-juan 2, Gao Shun-ji 1, Chen Zhong 1, Liu Zheng 1, Xu Ya-li 1 (1Department of Ultrasound, Second

Affiliate Hospital of Third Military Medical University, Chongqing 400037, China; 2

Department of Ultrasound,

the 302nd

Hospital of PLA, Beijing 100039, China)

Abstract

BACKGROUND: Neural stem cell transplantation is an emerging therapeutic option in the recovery of neural lesions and neurodegenerative diseases. Neural stem cell culture and differentiation lay a foundation for the further study.

OBJECTIVE: To improve the techniques for the isolation, cultivation, differentiation and identification of neural

stem cells, and to explore the biological characteristics of cells.

METHODS: The neural stem cells from C57BL/6 fetal rats were isolated and cultured in vitro using neurophere culture method followed by morphological and ultrastucture examination. The growth curve and cell cycle of passage 3 cells were drawn and analyzed. Nestin expression was tested by immunofluorescence. Neural stem cells induced in 1% and 10% fetal bovine serum were identified using anti-GFAP, anti-βIII -tubulin and anti-MBP by immunofluorescence.

RESULTS AND CONCLUSION: The neurospheres exhibited strong cell proliferation ability. Under transmission electron microscope, there was a high nuclear/cytoplasmic ratio in the neural stem cells, indicating a low differentiation degree. Immunofluorescence analysis revealed that neural stem cells were positive for Nestin.