Campbell Matthew, Humphries Peter
UCD School of Biomolecular Biomedical Sciences, Conway Institute, University College Dublin, Dublin, Ireland.
Methods Mol Biol. 2011;763:355-67. doi: 10.1007/978-1-61779-191-8_24.
Assessment of tight junction integrity in vitro is fundamental when studying molecular processes that may be implicated in barrier dysfunction. At the blood brain and inner blood retina barrier (BBB and iBRB, respectively) adjacent endothelial cells lining the microvasculature have been shown to have very low rates of fluid phase transcytosis and high electrical resistances, due in part to the expression of tight junction proteins at the apical periphery of these cells. While these high electrical resistances are difficult to achieve in vitro, owing to complex interactions of endothelial cells in vivo with astrocytes and pericytes, it is possible to make an assessment of paracellular permeability when cells are analysed on a number of different fronts. In this regard, we will outline here a method for determining trans-endothelial electrical resistance, tracer molecule diffusion, and tight junction protein localization in primary cultures of bovine retinal microvascular endothelial cells. This system allows for the screening of a wide range of pro- and anti-angiogenic molecules in an in vitro model of the iBRB and can accurately assess the role individual tight junction proteins play in maintaining tight junction integrity in response to various cell stimuli.
在研究可能与屏障功能障碍有关的分子过程时，体外评估紧密连接的完整性至关重要。在血脑屏障和血视网膜内屏障（分别为BBB和iBRB）中，微血管内衬的相邻内皮细胞已被证明具有非常低的液相转胞吞率和高电阻，部分原因是这些细胞顶端外周紧密连接蛋白的表达。虽然由于体内内皮细胞与星形胶质细胞和周细胞的复杂相互作用，在体外很难实现这些高电阻，但当在多个不同方面分析细胞时，可以对细胞旁通透性进行评估。在这方面，我们将在此概述一种用于确定牛视网膜微血管内皮细胞原代培养物中跨内皮电阻、示踪分子扩散和紧密连接蛋白定位的方法。该系统允许在iBRB的体外模型中筛选广泛的促血管生成和抗血管生成分子，并能准确评估单个紧密连接蛋白在响应各种细胞刺激时在维持紧密连接完整性中所起的作用。
