The impact of mechanical stresses induced by flow and stretch on barrier function in airway epithelial cells

流动和拉伸引起的机械应力对气道上皮细胞屏障功能的影响

• 批准号: RGPIN-2018-05591
• 负责人: Hirota, Jeremy
• 金额: $ 2.26万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=653618
• 关键词: impact; mechanical; stresses; induced; flow

Background: Much of the basic biology known about the airway epithelium has been deduced from static in vitro cell culture systems. Our understanding of airway epithelial cell biology may therefore be limited and under appreciate the impact of dynamic forces on biological properties important in epithelial barrier function. Our overarching objective is to incorporate physiological ranges of flow and stretch into our in vitro airway epithelial cell culture models and explore barrier function properties. ******Incorporation of flow into in vitro cell culture systems has showed that baseline mucus secretion is much higher than previously considered. Furthermore, incorporation of compressive stress showed that airway epithelial cells move within a monolayer and are not fixed in position. Incorporating flow and stretch into in vitro cell culture models of airway epithelial cells is likely to reveal unexplored biology. ******Aims: Use in vitro cell culture models to explore the impact of i) a physiological range of flows, ii) a physiological range of stretch, and iii) a combination of flow and stretch on airway epithelial cell barrier function******Methods: All three aims have experiments with outcome measurements that explore epithelial barrier function measured by transepithelial electrical resistance (TEER), molecular transport, wound repair, and gene expression. ******We will use a human airway epithelial cell line, Calu-3, to perform our experiments as this cell line creates tight mechanical barriers and has been validated for molecular transport studies. Experiments will be performed on cells grown under submerged monolayer and, where possible, air-liquid interface culture conditions. We will combine our cell cultures with a polydimethylsiloxane (PDMS) organ-on-a-chip device to allow integration of perfusion and stretch. For perfusion experiments, a syringe pump will be used to expose cells to a range of flow rates based on in vivo resting and peak flow rates. For stretch experiments, an adapted syringe pump system with an occlusion valve will be used to induce pressure changes that are transduced in vivo in response to stretch. To explore the potential for interactions between flow and stretch, we will combine these mechanical forces on our PDMS device.******Significance: Nascent work by others and our preliminary data suggest that mechanical forces induced by flow and stretch impact airway epithelial cell biology. Our proposed research program will explore unknown areas of airway epithelial cell biology by integrating physiological conditions currently absent from conventional cell culture systems, creating a foundation of knowledge for future basic and health research discoveries. Furthermore, our program will develop and validate technology and methods, protected by intellectual property, that can be adapted to other cell types to study how tissue specific mechanical forces impact function.

背景：许多关于气道上皮的基本生物学知识都是从静态体外细胞培养系统中推断出来的。因此，我们对气道上皮细胞生物学的理解可能是有限的，并且没有充分认识到动力对上皮屏障功能中重要的生物学特性的影响。我们的首要目标是将生理范围的流动和拉伸纳入我们的体外气道上皮细胞培养模型，并探索屏障功能特性。******将流体纳入体外细胞培养系统表明，基线粘液分泌比以前认为的要高得多。此外，压缩应力的结合表明气道上皮细胞在单层内移动，而不是固定的位置。在气道上皮细胞的体外细胞培养模型中加入流动和拉伸可能会揭示未知的生物学。******目的：使用体外细胞培养模型来探索i)生理范围的流动，ii)生理范围的拉伸，以及iii)流动和拉伸结合对气道上皮细胞屏障功能的影响******方法：所有三个目的都有实验结果测量，探索上皮屏障功能通过上皮电阻值（TEER），分子运输，伤口修复和基因表达测量。******我们将使用人类气道上皮细胞系Calu-3进行实验，因为该细胞系可产生紧密的机械屏障，并已在分子运输研究中得到验证。实验将在浸没单层和可能的气液界面培养条件下进行。我们将把我们的细胞培养与聚二甲基硅氧烷（PDMS）器官芯片设备相结合，以实现灌注和拉伸的整合。对于灌注实验，将使用注射泵将细胞暴露在基于体内静息和峰值流速的一系列流速中。对于拉伸实验，将使用一个带有闭塞阀的注射泵系统来诱导压力变化，这些压力变化在体内被传导以响应拉伸。为了探索流动和拉伸之间相互作用的潜力，我们将在PDMS设备上结合这些机械力。******意义：其他人的初步工作和我们的初步数据表明，由流动和拉伸引起的机械力会影响气道上皮细胞的生物学。我们提出的研究计划将通过整合目前传统细胞培养系统中缺乏的生理条件来探索气道上皮细胞生物学的未知领域，为未来的基础和健康研究发现奠定基础。此外，我们的项目将开发和验证受知识产权保护的技术和方法，这些技术和方法可以适用于其他细胞类型，以研究组织特定机械力如何影响功能。