海马神经干细胞不同传代方法的比较

中国组织工程研究与临床康复 第15卷 第6期 2011–02–05出版

Journal of Clinical Rehabilitative Tissue Engineering Research February 5, 2011 Vol.15, No.6

Department of Anatomy and Neurobiology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China

Li Mei ★, Studying for master’s degree, Department of Anatomy and Neurobiology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China limeihit@

Correspondence to: Xu Tie-jun, Department of

Anatomy and

Neurobiology, Xuzhou Medical

College, Xuzhou

221002, Jiangsu

Province, China

Supported by: the Natural Science

Foundation of Jiangsu Province,

No. BK2009087*; the Scientific Research

Innovation Project of

Postgraduate of

Higher Learning

Schools of Jiangsu Province, No.

CX10S-024Z *

Received: 2010-11-07 Accepted: 2010-12-06

徐州医学院解剖

学和神经生物学教研室，江苏省徐州市 221002

李梅★，女，1986年生，江苏省海安县人，汉族，徐州医学院在读硕士，主要从事神经干细胞的基础与临床应用研究。 limeihit@163. com

通讯作者：徐铁

军，徐州医学院解剖学和神经生物学教研室，江苏省徐州市 221002

中图分类号:R394.2 文献标识码:A

文章编号:1673-8225 (2011)06-00985-05

收稿日期：2010-11-07 修回日期：2010-12-06 (20101107008/W ·Q)

海马神经干细胞不同传代方法的比较**★

李 梅，王湘臻，徐铁军

Comparison of different passage methods for hippocampal neural stem cells

Li Mei, Wang Xiang-zhen, Xu Tie-jun Abstract

BACKGROUND: Neural stem cells (NSCs) cultured in vitro can form sphere due to its proliferation characteristics during suspension culture. How to isolate cell spheres into single cells is a problem facing us during subculture.

OBJECTIVE: To establish an ideal subculture method for harvesting NSCs from the rat hippocampus to obtain a large number of NSCs for investigation.

METHODS: NSCs were isolated from neonatal 1-day rat hippocampus. After a growth of typically 5-6 days, primary

neurospheres were passaged by mechanical dissociation, trypsin, TrypLE or Accutase digestion. Each method was used to

dissociate neurospheres for three passages every 7 days. The ratio of cell viability and neurosphere numbers were estimated on day 1 and day 4 respectively after passage. This experiment was repeated three times.

RESULTS AND CONCLUSION: Three enzymatic dissociation each obtained single-cell suspension; while mechanical dissociation resulted in mixtures of single cells and small neurospheres. The cell viability after Accutase treatment was

significantly higher than trypsin (P < 0.01) and TrypLE digestion (P < 0.05). Simultaneously, more newly formed neurospheres were generated by Accutase treatment as compared with other groups (P < 0.01). These indicated that in this experimental condition, enzymatic treatment using Accutase can be regarded as an ideal protocol for dissociating neurospheres into single cells with high cell survival which yield secondary neurospheres rapidly.

Li M, Wang XZ, Xu TJ. Comparison of different passage methods for hippocampal neural stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(6): 985-989. [ ]

摘要

背景：体外培养神经干细胞，在悬浮培养时由于自身增殖特性会形成球，传代时将会面临如何将细胞球分离成单细胞的问

题。 目的：寻求理想的大鼠海马神经干细胞传代方法，以获得大量可增生的神经干细胞以供研究。

方法：分离新生1 d SD 大鼠海马神经干细胞，原代培养至五六天时，分别用机械吹打法、胰蛋白酶、TrypLE 和Accutase 消化法分离神经干细胞球。之后每7 d 传代1次，连续传代3次。分别于每次传代后第1天和传代后第4天计数活细胞比例和细胞球数目，实验重复3次。

结果与结论：神经干细胞球经3种酶消化后获得的均是单细胞；经机械吹打后既有单个细胞，也有小细胞球分布于培养液中。在酶消化法中，Accutase 消化法传代后神经干细胞的活细胞比例明显高于胰蛋白酶消化(P < 0.01)和TrypLE 消化法 (P < 0.05)。同时，Accutase 消化法传代后新形成的细胞球数目也较其余各组多(P < 0.01)。提示在实验条件下，Accutase

消化法能够较好地将神经干细胞球分离成存活率较高、能快速形成新的克隆球的单个细胞，是较为理想的神经干细胞分离传代方法。

关键词：神经干细胞；海马；细胞培养；传代；免疫细胞化学；大鼠 doi:10.3969/j.issn.1673-8225.2011.06.009 李梅，王湘臻，徐铁军. 海马神经干细胞不同传代方法的比较[J].中国组织工程研究与临床康复，2011，15(6):985-989. [ ]

0 引言

利用神经干细胞移植治疗脑卒中、脊髓损伤和帕金森病等神经退行性病变是近年来神经科学领域研究的热点之一，其临床应用前景广阔[1-3]。但治疗过程中需要大量的神经干细胞，如何获得大量可增生的神经干细胞是研究其生物学性质和临床应用的基础。神经干细胞可由中枢神经系统的很多脑区原代培养获得[4-7]。神经干细胞在悬浮培养时由于自身增殖特性会形成球，当传代时将会面临如何将细胞球分离成单细胞问题。目前，细胞原代分离方法不同、培养条件不

一，关于传代方法也各不相同。虽然传代时将细胞分离成单个细胞看似一个简单的问题，但在实践中却常被分离成单个细胞的效果和细胞存活率的矛盾所困扰。为此，作者在原代培养出神经干细胞的基础上[8]，综合比较了常见的机械吹打法

[9-10]

、胰蛋白酶[11-12]、TrypLE 和Accutase

消化法等这些常见细胞解离和传代方法对传代后神经干细胞的存活和增殖等生物学性状的影响[13-15]，以建立一种有效的神经干细胞分离方法。

1 材料和方法

设计：细胞学体外观察，对比观察。