Richard L, Velasco P, Detmar M
Cutaneous Biology Research Center, Massachusetts General Hospital, Boston, USA.
Exp Cell Res. 1998 Apr 10;240(1):1-6. doi: 10.1006/excr.1998.3936.
Endothelial cells involved in tumor angiogenesis, wound healing, and inflammation are predominantly of microvascular origin and are functionally distinct from large vessel-derived endothelial cells which have been largely used for in vitro vascular research. To overcome the problems commonly involved in the culture of microvascular endothelial cells, including unreliable isolation techniques and low cell yields, we developed a simplified protocol for the selective cultivation of human dermal microvascular endothelial cells (HDMEC) obtained from neonatal foreskins, based on the transient, endothelial cell-specific induction of E-selection by tumor necrosis factor-alpha (TNF-alpha). Subconfluent primary cultures, consisting of a mixture of endothelial cells, fibroblasts, and keratinocytes, were treated with TNF-alpha for 6 h, and HDMEC were isolated by their selective binding to magnetic beads coupled with anti-E-selection monoclonal antibody. After two immunomagnetic purification steps, a homogenous population of HDMEC was obtained which showed typical cobblestone morphology, expressed CD31 and von Willebrand factor, proliferated in response to vascular endothelial growth factor, upregulated the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 in response to TNF-alpha, and formed capillary-like tubes in a three-dimensional collagen type I matrix. This simple technique may facilitate a more widespread use of microvascular endothelial cell cultures obtained from different human or animal organs for functional in vitro studies.
参与肿瘤血管生成、伤口愈合和炎症的内皮细胞主要起源于微血管，在功能上与主要用于体外血管研究的大血管来源的内皮细胞不同。为了克服微血管内皮细胞培养中常见的问题，包括分离技术不可靠和细胞产量低，我们基于肿瘤坏死因子-α（TNF-α）对E-选择素的短暂、内皮细胞特异性诱导，开发了一种简化方案，用于选择性培养从新生儿包皮获得的人真皮微血管内皮细胞（HDMEC）。将由内皮细胞、成纤维细胞和角质形成细胞混合组成的亚汇合原代培养物用TNF-α处理6小时，通过HDMEC与偶联抗E-选择素单克隆抗体的磁珠的选择性结合来分离HDMEC。经过两步免疫磁珠纯化后，获得了一群均一的HDMEC，其呈现典型的鹅卵石形态，表达CD31和血管性血友病因子，对血管内皮生长因子有增殖反应，对TNF-α有反应而上调细胞间黏附分子-1和血管黏附分子-1的表达，并在三维I型胶原基质中形成毛细血管样管。这种简单技术可能有助于更广泛地使用从不同人类或动物器官获得的微血管内皮细胞培养物进行功能性体外研究。
