Ando H, Kubin T, Schaper W, Schaper J
Department of Experimental Cardiology, Max Planck Institute, D-61231 Bad Nauheim, Germany.
Am J Physiol. 1999 May;276(5):H1755-68. doi: 10.1152/ajpheart.1999.276.5.H1755.
We established a culture system of porcine coronary microvascular endothelial cells (MVEC) with high cellular yield and purity >98%. Endothelial origin was confirmed by immunostaining, immunoblotting and fluorescence-activated cell sorter (FACS) analysis using low-density lipoprotein uptake, CD31, von Willebrand factor, and the lectin Dolichos biflorus agglutinin. MVEC were positive for alpha-smooth muscle actin in culture and in myocardium, as confirmed by FACS. Of the primary MVEC, approximately 30% expressed nitric oxide synthase (NOS) III in numbers decreasing from the first passage (6 +/- 1%) to the second passage (4 +/- 1%; P < 0.001 vs. primary isolates), whereas approximately 100% of aortic endothelial cells (AEC) expressed NOS III. In AEC, NOS III activity (pmol citrulline. mg protein-1. min-1) was 80 +/- 10 and was nearly abolished in the absence of calcium (5 +/- 1, P < 0.001). In primary MVEC, however, NOS III activity in the presence and absence of calcium was 20 +/- 4 and 25 +/- 5, respectively. We conclude that cardiac MVEC, in contrast to AEC, contain alpha-smooth muscle actin, show low-grade NOS III activity, and provide a suitable in vitro system for the study of endothelial pathophysiology.
我们建立了一种猪冠状动脉微血管内皮细胞（MVEC）培养体系，其细胞产量高且纯度>98%。通过免疫染色、免疫印迹以及使用低密度脂蛋白摄取、CD31、血管性血友病因子和双花扁豆凝集素进行荧光激活细胞分选（FACS）分析来确认内皮细胞来源。FACS证实，MVEC在培养物和心肌中α-平滑肌肌动蛋白呈阳性。在原代MVEC中，约30%表达一氧化氮合酶（NOS）III，其数量从第一代传代（6±1%）到第二代传代（4±1%；与原代分离物相比，P<0.001）逐渐减少，而约100%的主动脉内皮细胞（AEC）表达NOS III。在AEC中，NOS III活性（pmol瓜氨酸·mg蛋白-1·min-1）为80±10，在无钙情况下几乎完全消失（5±1，P<0.001）。然而，在原代MVEC中，有钙和无钙情况下的NOS III活性分别为20±4和25±5。我们得出结论，与AEC相比，心脏MVEC含有α-平滑肌肌动蛋白，显示低水平的NOS III活性，并为内皮病理生理学研究提供了一个合适的体外系统。
