College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China; College of Traditional Chinese Medicine and Pharmacology, Southwest University, Chongqing 400715, China; Chongqing Engineering Research Center for Pharmacological Evaluation, Chongqing 400715, China; Institute of Chinese Medicine, Southwest University, Chongqing 400715, China; Pharmacology of Chinese Materia Medica, TCM, Chongqing 400715, China.
Microvasc Res. 2013 Nov;90:199-205. doi: 10.1016/j.mvr.2013.08.004. Epub 2013 Aug 24.
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 2.2×10(7) 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 24h. 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.
脑微血管内皮细胞（BMECs）是血脑屏障的主要组成部分，在许多脑部疾病的发病机制中起着关键作用。BMEC 的原代培养已被用于各种体外脑血管疾病模型研究。然而，在原代大鼠 BMEC 的分离和培养中仍然存在几个问题，例如产量低、与其他细胞类型污染以及需要大量动物和昂贵的生长因子。在这项研究中，我们描述了一种简单、经济（无需任何生长因子）且可重复的方法，从新生大鼠大脑皮质中获得纯度（>99%）和产量（约 2.2×10(7)个/大鼠）较高的内皮细胞，主要来自灰质。体外检查确定，分离的细胞表达了典型的分化脑内皮细胞表型标志物，如多药耐药蛋白、血管性血友病因子、血小板内皮细胞黏附分子 1（PECAM-1/CD31）和细胞间黏附分子（ICAM）。这些细胞还具有通过相差显微镜和电子显微镜观察到的形态和超微结构特征。然后，分别使用 GFAP 和 α-SMA 鉴定潜在污染 BMEC 原代培养的星形胶质细胞和周细胞。并用 TNF-α 培养 24 小时，测试内皮细胞对外界刺激的特定反应。我们实验的所有这些结果表明，我们的方案提供了一种有效可靠的方法，可获得高纯度和高产量的大鼠 BMECs，并为研究细胞生理学、脑血管疾病、脑肿瘤、血脑屏障和神经血管单元等提供了有用的工具。
