Wills Tamara B, Heaney Allison M, Jane Wardrop K, Haldorson Gary J
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7060, USA.
Vet Clin Pathol. 2009 Dec;38(4):437-42. doi: 10.1111/j.1939-165X.2009.00152.x. Epub 2009 May 15.
Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells.
The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood.
Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti-CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146-positive cells were stained with fluorescein-conjugated Ulex europaeus agglutinin 1 (UEA-1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA-anticoagulated whole blood samples from 10 healthy client-owned dogs.
The anti-CD146-coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA-1-positive cells were obtained from whole blood, while >85-90% of cultured canine aortic endothelial cells were UEA-1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10-40 microm in size. Using immunomagnetic isolation, 43.4 +/- 15.6 CECs/mL (range 24-70/mL) were isolated from canine whole blood samples.
Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.
循环内皮细胞（CEC）浓度升高被认为是患有心血管疾病、肿瘤、血管炎、镰状细胞贫血、休克和败血症的人类患者血管损伤的生物标志物。免疫磁珠分离是目前用于计数人类CEC的技术，并且能够检测到少量细胞。
本研究的目的是确定免疫磁珠分离的标准方案是否可用于从犬全血中获取并计数CEC和内皮祖细胞（EPC）亚群。
用血管性血友病因子对培养的犬主动脉内皮细胞进行免疫组织化学染色，以验证形态和数量。使用与抗CD146偶联的磁珠，在培养物和犬全血中分离CEC/EPC。用异硫氰酸荧光素偶联的荆豆凝集素1（UEA-1）对CD146阳性细胞进行染色，以确认内皮来源，并使用荧光显微镜手动计数细胞。然后将该方法应用于来自10只健康客户拥有犬的乙二胺四乙酸抗凝全血样本。
抗CD146包被的磁珠（>5个/细胞）与培养的犬主动脉内皮细胞结合。从全血中仅获得罕见的UEA-1阳性细胞，而>85-90%的培养犬主动脉内皮细胞为UEA-1阳性。培养的犬主动脉内皮细胞回收率>86%。犬全血中的CEC表面附着有>8个磁珠，大小为10-40微米。使用免疫磁珠分离，从犬全血样本中分离出43.4±15.6个CEC/mL（范围为24-70个/mL）。
免疫磁珠分离是全血中计数犬CEC/EPC的可接受方法。有必要进一步研究评估不同犬病中CEC/EPC浓度的临床意义。
