Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil.
Methods Mol Biol. 2022;2409:207-222. doi: 10.1007/978-1-0716-1879-0_14.
This chapter will discuss reliable and relatively easy and fast strategies to evaluate the integrity of endothelial cell monolayers when infected by dengue virus (DENV). Human brain microvascular endothelial cells (HBMEC) were exploited here as general model of vessel wall core, but it may also be used as an in vitro simplified model of blood brain barrier (BBB). The integrity of endothelial cells monolayer can be inferred using a transwell culture system by: (1) measuring transendothelial electrical resistance (TEER) using a Voltohmmeter; (2) analyzing the monolayer permeability to fluorescent-conjugated proteins and fluorimetric assay; (3) investigating virus extravasation by quantitative RT-PCR and plaque conventional assay. The rational to use those strategies is that vascular alterations are often observed during dengue infection, being associated to disease severity. The vasculature core consists of a barrier of endothelial cells, which are tightly adhered by the expression of adhesion molecules and tight junctions. This structure must be preserved in order to control the flux of cells and metabolites from the circulation to the tissues and to maintain vascular homeostasis. Therefore, experimental assays that allow evaluation of endothelial integrity can be useful platforms to further understand disease pathogenesis and screen pharmaceutical interventions to control vascular disturbance.
这一章将讨论可靠且相对简单快速的策略，以评估感染登革热病毒（DENV）后的内皮细胞单层的完整性。人类脑微血管内皮细胞（HBMEC）在这里被用作血管壁核心的一般模型，但也可以用作血脑屏障（BBB）的体外简化模型。通过以下方法可以推断内皮细胞单层的完整性：（1）使用伏特计测量跨内皮电阻（TEER）；（2）通过荧光标记蛋白和荧光测定分析单层通透性；（3）通过定量 RT-PCR 和斑块常规测定法研究病毒外渗。使用这些策略的原因是，在登革热感染期间经常观察到血管改变，与疾病严重程度相关。血管核心由内皮细胞组成的屏障组成，这些细胞通过粘附分子和紧密连接的表达紧密附着。为了控制细胞和代谢物从循环到组织的通量并维持血管内环境稳定，必须保持这种结构。因此，允许评估内皮完整性的实验测定可以成为进一步了解疾病发病机制和筛选控制血管紊乱的药物干预的有用平台。
