Yuan Ke, Orcholski Mark E, Huang Ngan F, de Jesus Perez Vinicio A
Division of Pulmonary and Critical Care Medicine, School of Medicine, Stanford University; Stanford Cardiovascular Institute, School of Medicine, Stanford University.
Stanford Cardiovascular Institute, School of Medicine, Stanford University; VA Palo Alto Health Care System, Department of Cardiothoracic Surgery, School of Medicine, Stanford University.
J Vis Exp. 2016 Dec 19(118):54617. doi: 10.3791/54617.
Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.
血管生成是指从现有血管形成新血管的过程。新血管的生长需要内皮细胞协调增殖、迁移和排列以形成管状结构，随后募集周细胞以提供壁层支持并促进血管成熟。当前的体外细胞培养方法无法完全重现内皮细胞和周细胞相互作用以产生功能性血管的复杂生物环境。我们展示了体内基质胶栓试验的一种新应用，即使用严重联合免疫缺陷突变（SCID）小鼠来研究内皮细胞与周细胞的相互作用以及功能性血管的形成。简而言之，将基质胶与含有内皮细胞（有或无周细胞）的溶液混合，然后注射到麻醉的SCID小鼠背部。14天后，取出基质胶栓，固定并切片进行组织学分析。使用商业统计平台可以对成熟血管（由管腔内存在红细胞定义）的周细胞覆盖长度、数量、大小和范围进行量化并在实验组之间进行比较。除了用作血管生成试验外，这种基质胶栓试验还可用于进行基因研究并作为药物发现的平台。总之，该方案将使研究人员能够补充现有的体外试验，以研究内皮细胞与周细胞的相互作用及其与全身或肺血管生成的相关性。
