Nakatsu Martin N, Hughes Christopher C W
Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, California, USA.
Methods Enzymol. 2008;443:65-82. doi: 10.1016/S0076-6879(08)02004-1.
Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a multistage process in which activated endothelial cells (EC) degrade basement membrane, sprout from the parent vessel, migrate, proliferate, align, undergo tube formation, and eventually branch and anastomose with adjacent vessels. Here we describe a three-dimensional in vitro assay that reproduces each of these steps. Human umbilical vein endothelial cells (HUVEC) are cultured on microcarrier beads, which are then embedded in a fibrin gel. Fibroblasts cultured on top of the gel provide factors that synergize with bFGF and VEGF to promote optimal sprouting and tube formation. Sprouts appear around day 2, lumen formation begins at day 4, and at day 10 an extensive anastomosing network of capillary-like tubes is established. The EC express a similar complement of genes as angiogenic EC in vivo and undergo identical morphologic changes during tube formation. This model, therefore, recapitulates in vivo angiogenesis in several critical aspects and provides a system that is easy to manipulate genetically, can be visualized in real time, and allows for easy purification of angiogenic EC for downstream analysis.
血管生成是指从现有脉管系统形成新的血管。这是一个多阶段过程，在此过程中，活化的内皮细胞（EC）降解基底膜，从母血管发芽，迁移、增殖、排列，形成管腔，最终分支并与相邻血管吻合。在此，我们描述了一种三维体外试验，该试验重现了这些步骤中的每一步。人脐静脉内皮细胞（HUVEC）在微载体珠上培养，然后将微载体珠包埋在纤维蛋白凝胶中。在凝胶顶部培养的成纤维细胞提供与碱性成纤维细胞生长因子（bFGF）和血管内皮生长因子（VEGF）协同作用的因子，以促进最佳的发芽和管腔形成。芽在第2天左右出现，管腔形成在第4天开始，在第10天建立起广泛的类似毛细血管的吻合网络。这些内皮细胞表达的基因与体内血管生成内皮细胞的基因互补相似，并且在管腔形成过程中经历相同的形态变化。因此，该模型在几个关键方面重现了体内血管生成，并提供了一个易于进行基因操作、可实时可视化且便于纯化血管生成内皮细胞以进行下游分析的系统。
