Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy
Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy
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DOI：
10.1038/pr.2017.28
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发表时间：
2017-06-01
影响因子：
3.6
通讯作者：
Hosoya, Mitsuaki
中科院分区：
文献类型：
--
作者：
Miyazaki, Kyohei;Hashimoto, Koichi;Hosoya, Mitsuaki
背景：病毒相关性急性脑病（VAE）是儿童常见病毒感染引起的严重中枢神经系统并发症。 VAE 的病理生理学被认为是内皮损伤。本研究旨在建立体外VAE模型，用于评估促炎细胞因子TNF-α引起的内皮损伤。方法：将Transwell生长的人脐静脉内皮细胞（HUVEC）单层与连续稀释的TNF-α一起孵育。使用阻抗谱测量跨内皮电阻（TER）。 TNF-α 处理后 HUVEC 的通透性变化由异硫氰酸荧光素 (FITC) 缀合的葡聚糖测定。此外，通过免疫荧光染色观察TNF-α诱导的claudin-5形态学变化和细胞凋亡。结果：TER的减少、TER恢复到基线的时间以及通透性的增加均依赖于TNF-α浓度。免疫荧光染色显示，在 TNF-α 处理后，claudin-5 以剂量依赖性方式离域。此外，在高TNF-α浓度下观察到一些凋亡细胞。结论：TER测量与渗透性测定相结合可用于评估体外模型中血管内皮细胞的渗透性。这些评估方法将有助于开发专注于血管通透性的具体治疗方法，以及寻找 VAE 治疗的新治疗策略。
BACKGROUND: Virus-associated acute encephalopathy (VAE) is a severe central nervous system complication caused by common viral infections in children. The pathophysiology of VAE is thought to be endothelial injury. This study was designed to establish an in vitro VAE model for evaluating endothelial injury caused by the proinflammatory cytokine TNF-alpha.METHODS: Transwell-grown human umbilical vein endothelial cells (HUVECs) monolayers were incubated with serially diluted TNF-alpha. Transendothelial electrical resistance (TER) was measured using impedance spectroscopy. Permeability changes of HUVECs after TNF-alpha treatment were determined by fluorescein isothiocyanate (FITC)-conjugated dextran. Moreover, TNF-alpha-induced morphological changes in claudin-5 and apoptosis were observed by immunofluorescent staining.RESULTS: The decrease in TER, time of TER recovery to baseline, and increase in permeability were all dependent on TNF-alpha concentration. Immunofluorescent staining showed that claudin-5 was delocalized after TNF-alpha treatment in a dose-dependent manner. ln addition, some apoptotic cells were observed at high TNF-alpha concentrations.CONCLUSION: TER measurement combined with a permeability assay could be useful for evaluating vascular endothelial cell permeability in an in vitro model. These evaluation methods will contribute to both the development of specific treatments focusing on vascular permeability, and the search for a novel therapeutic strategy in VAE treatment.