Department of Kinesiology and Applied Physiology, College of Health Sciences, University of Delaware.
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago.
J Vis Exp. 2022 Jun 10(184). doi: 10.3791/63999.
Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45- cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.
血管内皮细胞排列在血管系统的壁上，在多种生理过程中发挥重要作用，包括血管张力调节、屏障功能和血管生成。内皮细胞功能障碍是严重心血管疾病进展的标志性预测因子和主要驱动因素，但潜在机制仍知之甚少。能够以天然形式从各种血管床分离和分析内皮细胞，将深入了解心血管疾病的发生过程。本方案介绍了从小鼠皮下和肠系膜脂肪组织中分离其动脉血管系统的操作流程。然后，使用专门的消化酶混合物消化分离的动脉，该混合物专注于释放具有功能活性的内皮细胞。通过流式细胞术分析，使用 CD31+/CD45-细胞作为阳性内皮细胞鉴定的标志物，评估消化后的组织。细胞可以立即进行下游功能测定，或用于生成原代细胞系。从不同血管床分离和消化动脉的技术将为研究人员提供从感兴趣的动脉中评估新鲜分离的血管细胞的选择，并允许他们对特定细胞类型进行广泛的功能测试。
