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胰蛋白酶消化强度优化获得高纯度体外培养的星形胶质细胞

靳辉, 杨蓬勃, 冯改丰, 贾宁, 杨维娜, 王唯析^*

西安交通大学医学院人体解剖与组织胚胎学系，西安，710061

摘要

本文旨在观察胰蛋白酶消化对体外培养的星形胶质细胞纯度的影响，优化星形胶质细胞培养方法。常规分离新生SD大鼠大脑皮质，分别用0.25% 胰蛋白酶消化20、30和40 min制备单细胞悬液并接种细胞。细胞长满瓶底时进行恒温摇床振荡，各组再分为常规消化的对照组和二次胰蛋白酶消化组进行传代纯化。倒置相差显微镜观察细胞生长情况，MTT法检测细胞增殖，GFAP免疫荧光分析星形胶质细胞纯度并观察其形态，流式细胞术分析细胞凋亡。结果显示，胰蛋白酶消化20 min的细胞在原代培养9 d长满瓶底。在本研究中，延长胰蛋白酶消化时间至30 min，细胞增殖更快，培养7 d即可铺满瓶底，且星形胶质细胞形态正常，纯度达(70.2 ± 4.0)%，较20 min组有显著性提高（P < 0.01）。40 min组虽然星形胶质细胞纯度也较20 min组有所提高，但细胞增殖缓慢且损伤明显。在恒温摇床振荡结束后，进行二次胰蛋白酶消化可减少传代后杂细胞数量。二次胰蛋白酶消化组第一代（P1）的GFAP阳性率普遍高于对照组，其中30 min+二次胰蛋白酶消化组GFAP阳性率最高，达到(98.1 ± 1.7)%，相当于20 min+对照组P3水平，且二者的凋亡率无显著性差异。以上结果表明，胰蛋白酶消化30 min+二次消化能有效提高星形胶质细胞纯度，缩短原代培养及纯化时间，是体外快速获得高纯度星形胶质细胞的有效方法。

关键词： 星形胶质细胞; 细胞培养; 胰蛋白酶

分类号：Q25

[Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes.] [Article in Chinese]

JIN Hui, Yang Peng-Bo, FENG Gai-Feng, Jia Ning, Yang Wei-Na, WANG Wei-Xi^*

Department of Human Anatomy and Histo/Embryology, Xi’an ]iaotong University College of Medicine, Xi'an 710061,China

Abstract

To observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn SD rats was isolated and digested with 0.25% typsin for 20, 30, and 40 min, respectively. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each digestive group was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptosis rate of purified astrocytes. As a result, the cells being digested for 20 min usually reached confluence at 9 d after seeding. In this study, when the digestion time was extended to 30 min, cells grew faster and reached confluence at 7 d after seeding. At the same time, the morphology of astrocytes was normal and the GFAP positive rate reached (70.2 ± 4.0)% which was much higher than that of the 20 min group (P < 0.01). Compared to 20 min group, the GFAP positive rate was higher, but the cell proliferation was slower and cell injury was more obvious in 40 min group. After shaking at a constant temperature, two times of trysine digestion could decrease the number of contaminated cells after passage. The GFAP positive rate of two-time-digestion groups in passage 1 (P1) was higher than that of the control groups, and 30 min+ two-time-digestion group reached the highest level, about (98.1 ± 1.7)%, which was equivalent to that of the 20 min+control group in P3. However, the apoptosis rate in these two groups showed no significant difference. From above results, we could conclude that 30 min+two-time of trysine digestion effectively improved the purity of astrocytes and shortened the time of primary culture and purification. It would be a rapid and effective method to obtain astrocytes with high purity in vitro.

Key words: astrocytes; cell culture; typsin

收稿日期：2014-07-23　　录用日期：2014-10-30

通讯作者：王唯析　　E-mail: wangwx@mail.xjtu.edu.cn

引用本文：

靳辉, 杨蓬勃, 冯改丰, 贾宁, 杨维娜, 王唯析. 胰蛋白酶消化强度优化获得高纯度体外培养的星形胶质细胞[J]. 生理学报 2015; 67 (1): 103-109.

JIN Hui, Yang Peng-Bo, FENG Gai-Feng, Jia Ning, Yang Wei-Na, WANG Wei-Xi. [Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes.] [Article in Chinese]. Acta Physiol Sin 2015; 67 (1): 103-109 (in Chinese with English abstract).