Salter Margaret M, Seeto Wen J, DeWitt Blake B, Hashimi Sarah A, Schwartz Dean D, Lipke Elizabeth A, Wooldridge Anne A
Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
Am J Vet Res. 2015 Feb;76(2):174-87. doi: 10.2460/ajvr.76.2.174.
To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses.
Jugular venous blood samples from 24 adult horses.
Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates.
Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry.
ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.
从马的外周血样本中分离并鉴定内皮祖细胞集落形成细胞（ECFCs；内皮祖细胞的一种亚型）。
来自24匹成年马的颈静脉血样本。
将血样本在内皮细胞生长培养基中培养。通过体外荧光标记乙酰化低密度脂蛋白（DiI-Ac-LDL）摄取功能测定和血管小管形成功能测定对分离出的ECFCs进行鉴定。通过间接免疫荧光测定和流式细胞术评估内皮细胞（CD34、CD105、血管内皮生长因子受体2和血管性血友病因子）和造血细胞（CD14）标志物的表达。通过对DiI-Ac-LDL摄取、血管小管形成和细胞倍增率的系列评估来确定衰老前的传代次数。
来自3匹马的样本在12±2.5天产生集落，具有特征性的内皮单层鹅卵石形态，扩增时大量生长。马ECFCs在体外形成血管小管，并摄取DiI-Ac-LDL（74.9±14.7%阳性细胞）。到第5代时，小管形成和DiI-Ac-LDL摄取减少。免疫荧光测定显示马ECFCs对血管性血友病因子、血管内皮生长因子受体2、CD34和CD105呈阳性，流式细胞术显示对CD14和CD105呈阳性。
ECFCs可从马的外周血中分离出来，其特征与其他物种描述的相似。这些细胞可能在与缺血或血管化延迟相关的马疾病中具有潜在的治疗用途。
