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A simple method for isolating and culturing the rat brain microvascular endothelial cells

细胞生物学 川地31 血管性血友病因子 内皮 血脑屏障 内皮干细胞 周细胞 体外 生物 星形胶质细胞 细胞培养 病理 血小板 免疫学 神经科学 医学 中枢神经系统 生物化学 内分泌学 遗传学
作者
Yang Liu,Qiang Xue,Qing Tang,Min Hou,Hongyi Qi,Gang Chen,Weihai Chen,Jifen Zhang,Yi Chen,Xiaoyu Xu
出处
期刊:Microvascular Research [Elsevier]
卷期号:90: 199-205 被引量:48
标识
DOI:10.1016/j.mvr.2013.08.004
摘要

Brain microvascular endothelial cells (BMECs), a main component of the blood–brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (> 99%) and yield (about 2.2 × 107 per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24 h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood–brain barrier and neurovascular units, etc.
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