Johnson Edward K, Schelling Margaret E, Quitadamo Ian J, Andrew Shayne, Johnson Emily C
School of Molecular Biosciences, College of Education, Washington State University, Pullman, 99164, USA.
Microvasc Res. 2002 Sep;64(2):278-88. doi: 10.1006/mvre.2002.2423.
Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO(4), and 500 microM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low-density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelial-specific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for alpha-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern.
冠状动脉微血管内皮细胞（CMECs）在许多生理过程中发挥着重要作用。来自大型猪的猪CMECs已被分离并成功培养。然而，由于小型猪比大型猪具有研究优势，小型猪CMEC（MPCMEC）培养物可能作为心血管研究的体外猪模型的替代物。我们使用一种简化技术从6只帕内平托小型猪中分离出MPCMECs，并开发了一种使其成功培养的系统。通过使用无菌技术获得的左心室样本经胶原酶消化来分离MPCMECs。MPCMECs的原代分离物在补充有20%胎牛血清、4 mM硫酸镁和500 microM二丁酰环磷腺苷的完全DMEM中稳定生长，并在7 - 10天内达到汇合。通过对乙酰化低密度脂蛋白的快速（4小时）摄取、对血小板/内皮细胞黏附分子-1（PECAM-1，CD31）、血管性血友病因子（vWf）相关抗原、血管内皮钙黏蛋白（VE-钙黏蛋白）、内皮型一氧化氮合酶（eNOS）的免疫染色以及使用两种异硫氰酸荧光素标记的内皮特异性凝集素（双花扁豆凝集素和荆豆凝集素-1）的阳性染色来证明其内皮来源。MPCMECs对α-平滑肌肌动蛋白也表现出免疫染色。MPCMECs在没有二丁酰环磷腺苷的情况下成功传代培养，并在传至第6代时继续表达PECAM-1和vWf，但不表达eNOS。亚汇合MPCMECs的典型形态由呈漩涡状、人字形生长的细长细胞组成。
