Purity, Cell Viability, Expression of GFAP and Bystin in Astrocytes Cultured by Different Procedures
Purity, Cell Viability, Expression of GFAP and Bystin in Astrocytes Cultured by Different Procedures
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DOI：
10.1002/jcb.22375
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发表时间：
2010-01-01
影响因子：
4
通讯作者：
Ke, Ya
中科院分区：
文献类型：
--
作者：
Du, Fang;Qian, Zhong Ming;Ke, Ya
原代星形胶质细胞培养是神经生物学研究中最常用的体外模型。我们推测，不同的协议可能会导致差异不仅在星形胶质细胞的百分比，但也在他们的生物学特性。本研究采用MTT法、免疫细胞化学染色和Western blot分析等方法，研究了四种主要的体外培养方法对星形胶质细胞纯度、细胞活力、胶质细胞酸性蛋白（GFAP）和bystin表达的影响。我们证明，通过传代培养（SC）程序产生的星形胶质细胞的纯度（98.9%）显著高于原代培养（PC），振荡一次培养（SK-1）和振荡两次培养（SK-2）。我们还表明，通过SK-2或SK-1程序纯化的星形胶质细胞中GFAP和bystin的表达显著高于通过PC或SC制备的星形胶质细胞中的表达。此外，SK-2或SK-1培养的星形胶质细胞在缺血后大多数时间点的细胞存活率均高于PC或SC培养的星形胶质细胞，这表明“震荡”诱导的物理刺激可能是星形胶质细胞缺血再灌注损伤的重要原因。或培养操作可能能够激活星形胶质细胞，这意味着不同的程序不仅诱导星形胶质细胞的纯度不同，而且在生物学特性上也存在差异，如激活的星形胶质细胞的百分比，GFAP和bystin表达以及对缺血的反应。因此，有必要对“培养方案因素”对星形胶质细胞生物学特性的影响进行更详细的分析。J.细胞。109：30-37，2010. (C)2009 Wiley-Liss，Inc.
Primary astrocyte cultures are the most commonly used in vitro model For neurobiological studies. We speculated that different protocols might induce differences not only in the percentage of astrocytes but also in their biological characteristics. In this study, we investigated the effects of four major protocols on the purity of astrocytes, cell viability, expression of glial fibrillary acidic protein (GFAP) and bystin of cultured astrocytes using MTT assay, immunocytochemical staining, and Western blot analysis. We demonstrated that the purity of astrocytes (98.9%) generated by the subculture (SC) procedure is significantly higher than those generated by primary culture (PC), shaken once culture (SK-1) or shaken twice culture (SK-2). We also showed that expressions of GFAP and bystin in astrocytes that are purified by the SK-2 or SK-1 procedures are significantly higher than those in astrocytes prepared by PC or SC. In addition, astrocytes cultured by SK-2 or SK-1 have a higher level of cell viabilities at most time points after ischemia compared with astrocytes cultured by PC or SC. These suggested that physical stimulation induced by "shaken" or culture operation might be able to activate astrocytes and implied that different procedures induce differences not only in the purity but also in the biological characteristics of astrocytes, such as the percentage of activated astrocytes, GFAP, and bystin expressions and responses to ischemia. A more detailed analysis about the effect of "culture protocol factor" on the biological characteristics of astrocytes is absolutely needed. J. Cell. Biochem. 109: 30-37, 2010. (C) 2009 Wiley-Liss, Inc.