Isolation and characterization of large numbers of endothelial cells for studies of cell signaling.

Studies of endothelial cell signaling involving cholesterol-rich domains require large numbers of cells of bona fide origin. The growth of any cell in culture, particularly for extended periods, results in an altered phenotype that could include changes in the properties of caveolae and lipid raft structures. While continuously propagated cells are used to study specific questions because their origin is known and because proteins of interest are still expressed, such reasoning is an oversimplification and can lead to findings that are descriptive of the cell's adaptation to culture rather than its original phenotype. We are particularly cognizant of this concern as we examine caveolar signaling domains in endothelial cells. Here we present a reproducible method for the isolation and characterization of large numbers of bona fide endothelial cells suitable for studies of the regulation of receptor signal transduction. Digestion of guinea pig hearts with collagenase results in the liberation of cells that adhere to collagen-coated plastic and express platelet-endothelial cell adhesion molecule 1 (PECAM-1) and binding sites for Ulex europaeus agglutinin 1 (UEA-1) that permit segregation of cells using fluorescence-activated cell sorting. Growth of cells over 7 doublings results in enrichment in the expression of both PECAM-1 and UEA-1 and retention of functional low-density lipoprotein receptor. The ability of cells to differentiate into endothelial tubes at any stage during their characterization up to 20 doublings in culture suggests that this method can be employed to generate endothelial cells that are minimally altered from their site of origin.

涉及富含胆固醇结构域的内皮细胞信号传导研究需要大量真正来源的细胞。任何细胞在培养中的生长，尤其是长时间培养，都会导致表型改变，这可能包括小窝和脂筏结构特性的变化。虽然连续传代的细胞因其来源已知且感兴趣的蛋白质仍有表达而被用于研究特定问题，但这种推理过于简单化，可能导致所得到的结果是描述细胞对培养环境的适应，而非其原始表型。在我们研究内皮细胞的小窝信号传导结构域时，我们特别意识到这一问题。在此，我们介绍一种可重复的方法，用于分离和鉴定大量适合研究受体信号转导调控的真正内皮细胞。用胶原酶消化豚鼠心脏可释放出能黏附于胶原蛋白包被的塑料培养皿上的细胞，这些细胞表达血小板 - 内皮细胞黏附分子1（PECAM - 1）以及欧洲荆豆凝集素1（UEA - 1）的结合位点，从而可利用荧光激活细胞分选技术对细胞进行分离。经过7次以上倍增的细胞生长会使PECAM - 1和UEA - 1的表达增加，并保留功能性低密度脂蛋白受体。在培养过程中直至20次倍增的鉴定过程中的任何阶段，细胞分化为内皮管的能力表明，该方法可用于产生与其来源部位相比变化极小的内皮细胞。