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树鼩脑星形胶质细胞的体外培养及纯化

龚敏, 李树清^*, 李凡

昆明医学院病理生理学教研室，昆明 650031

摘要

本文旨在建立低等灵长类动物树鼩(Tupaia belangeri)脑星形胶质细胞(astrocyte, AS)原代培养及纯化的技术，为利用新型实验动物树鼩进行研究工作而建立体外模型。将新生树鼩大脑皮质机械分离，置于4°C冰箱20 min以损伤神经元，皮质组织块用胰蛋白酶消化后制成细胞悬液，分次贴壁去除成纤维细胞，培养的混合细胞在每次换液时用0.005%胰蛋白酶轻柔漂洗去除神经元。细胞长满培养瓶底面积约70%时，用0.025%胰酶溶液静置消化，至肉眼可见一层白色薄膜从瓶底脱落时终止消化，此白色薄膜即为AS层。AS传至第三代时，用抗胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)抗体进行免疫组织化学染色和免疫荧光染色鉴定。结果显示，本方法所得的树鼩脑AS的纯度可达98%以上。该结果提示，这种通过分次贴壁法结合差异消化的培养及纯化技术可获得高纯度的树鼩脑AS，为建立神经系统疾病新的体外细胞培养模型打下了基础。

关键词： 树鼩; 形胶质细胞; 纯化; 免疫组织化学; 免疫荧光

分类号：R332

[Primary culture and purification of cerebral astrocyte of tree shrew.] [Article in Chinese]

GONG Min, LI Shu-Qing^*, LI Fan

Department of Pathophysiology, Kunming Medical College, Kunming 650031, China

Abstract

The aim of this study is to establish a method to culture and purify cerebral astrocyte of tree shrew (Tupaia belangeri), a kind of new laboratorial animal which is a relative of primates. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and placed in 4°C for 20 min to injure neurons. The cortical tissue was disaggregated by trypsin digestion. Differential attachment method was used to remove fibroblasts. The mixed culture was rinsed by trypsin (0.005%) solution to remove neurons. Upon reaching 70% confluence, the culture was subjected to static trypsin digestion until a white slice film exfoliated from the bottom of culture bottle. This film, i.e. astrocyte layer, was taken out and cultured, and the third passage was identified by immunocytochemical staining and immunofluorescence with anti-glial fibrillary acidic protein (GFAP) antibody. The result showed the purity of tree shrew astrocytes was more than 98%. Thus the method to culture highly purified astrocyte of tree shrew was successfully established, which would contribute to further study in central nervous system physiology and diseases in this new laboratorial animal.

Key words: tree shrew; astrocyte; purification; immunohistochemistry; immunofluorescence

收稿日期：2010-06-25　　录用日期：2010-12-15

通讯作者：李树清　　E-mail: shuqing591@hotmail.com

引用本文：

龚敏, 李树清, 李凡. 树鼩脑星形胶质细胞的体外培养及纯化[J]. 生理学报 2011; 63 (1): 89-92.

GONG Min, LI Shu-Qing, LI Fan. [Primary culture and purification of cerebral astrocyte of tree shrew.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (1): 89-92 (in Chinese with English abstract).