Core B - Human Cell/Tissue Acquisition and Physiology Care

项目概要 核心 B 更新的主要目标是提供体外和离体人体平台来研究机制 调节人气道平滑肌（HASM）的兴奋-收缩（EC）耦合。核心B的目标 是建立、表征并提供原代 HASM 细胞，生成单个 HASM 细胞测量结果 细胞骨架 (CSK) 刚度和力生成（磁扭转细胞术 – MTC），并执行人类 所有项目和核心的精密切割肺切片 (hPCLS) 研究。我们的实验室拥有超过 25 年的经验 原代 HASM 细胞的建立、培养和表征。核心 B 还将提供哮喘- 和非哮喘衍生的 HASM 来表征疾病状态产生的内在差异并捕获 人类反应的异质性。 hPCLS 将作为测量生理相关性的平台 完整人体组织中的支气管收缩和扩张。此外，我们将使用细胞因子和肥大细胞暴露 模拟 T2 炎症环境的模型。目标1将提供HASM细胞的新型细胞模型 高反应性以探索 EC 耦合、单细胞缩短和力之间的关系 产生哮喘中的 GPCR 激动剂和拮抗剂。项目 1 将使用这些模型来研究 TGF-β1 调节气道收缩力的机制；项目 2 和 4 将阐明机制 调节 TAS2R 和 OGR1 介导的 HASM 松弛；项目3将开发靶向疗法 减弱 HASM 的收缩性并增强其松弛。目标 2 将提供一个集成的组织模型 HASM 收缩和气道高反应性 (AHR)，用于研究 GPCR 信号传导。使用 hPCLS、项目 1 将检查 TGF-β1 调节小气道收缩和扩张的机制；项目 2 和 4 将阐明 TAS2R 和 ORG1 介导小气道扩张的机制；项目 3 将 发现消除 AHR 并通过靶向治疗增强支气管扩张的新方法。目标3将 解决炎症介质改变 GPCR 功能和支气管收缩的机制 在 hPCLS 中。利用这些平台，项目 1 将解决细胞因子如何调节 TGF-β1 对气道的影响 收缩性；项目 2 和 4 将描述 TAS2R 和 OGR1 介导的支气管扩张在 细胞因子暴露或肥大细胞脱粒的背景；项目 3 将开发能够减弱 尽管 T2 炎症，AHR 并恢复支气管扩张剂低反应性。核心B的贡献是 意义重大，因为我们的模型将阐明消除 AHR 并增强支气管扩张的分子靶点 哮喘的背景。 Core B 的创新专注于最先进的单细胞力测量 使用体外和离体模型的 HASM 的生成和小气道功能。此外，我们的模型仅使用 人类细胞和组织减轻了 ASM 中 EC 耦合研究的物种差异。一个 擅长研究 HASM 细胞和组织的集中人体细胞和组织核心是一项独特的资产，将 为整个计划项目提供质量保证。

Project Summary The principal goal of the Core B renewal is to provide in vitro and ex vivo human platforms to study mechanisms regulating excitation-contraction (EC) coupling in human airway smooth muscle (HASM). The objective of Core B is to establish, characterize and provide primary HASM cells, generate single HASM cell measurements of cytoskeletal (CSK) stiffness and force generation (magnetic twisting cytometry – MTC), and perform human precision cut lung slice (hPCLS) studies for all Projects and Cores. Our laboratory has over 25 years of experience in the establishment, cultivation, and characterization of primary HASM cells. Core B will also provide asthma- and non-asthma-derived HASM to characterize intrinsic differences engendered by disease-state and to capture heterogeneity of human responses. hPCLS will serve as the platform to measure physiologically relevant bronchoconstriction and dilation in intact human tissue. Additionally, we will use cytokine- and mast cell-exposure models to mimic a T2 inflammatory milieu. Aim 1 will provide novel cellular models of HASM cell hyperresponsiveness to explore the relationship among EC coupling, single cell shortening, and force generation to GPCR agonists and antagonists in asthma. Using these models, Project 1 will study mechanisms underlying TGF-β1 modulation of airway contractility; Projects 2 and 4 will elucidate mechanisms modulating TAS2R- and OGR1-mediated HASM relaxation; and Project 3 will develop targeted therapeutics to attenuate contractility and enhance relaxation of HASM. Aim 2 will provide an integrated tissue model of HASM contraction and airway hyperresponsiveness (AHR) to study GPCR signaling. Using hPCLS, Project 1 will examine mechanisms by which TGF-β1 modulates constriction and dilation of small airways; Projects 2 and 4 will elucidate mechanisms by which TAS2R and ORG1 mediate dilation of small airways; and Project 3 will discover novel ways to abrogate AHR and enhance bronchodilation with targeted therapeutics. Aim 3 will address the mechanisms by which inflammatory mediators alter GPCR function and bronchoconstriction in hPCLS. Using these platforms, Project 1 will address how cytokines modulate TGF-β1 effects on airway contractility; Projects 2 and 4 will characterize the roles of TAS2R- and OGR1-mediated bronchodilation in the context of a cytokine exposure or mast cell degranulation; and Project 3 will develop ligands that will attenuate AHR and restore bronchodilator hyporesponsiveness despite T2 inflammation. The contribution of Core B is significant as our models will elucidate molecular targets to abrogate AHR and enhance bronchodilation in the context of asthma. The innovation of Core B focuses on the state-of-the-art measurements of single cell force generation and small airway function of HASM using in vitro and ex vivo models. Further our models use only human cells and tissue mitigating species differences that confounds the study of EC coupling in ASM. A centralized human cell and tissue core adept in the study of HASM cells and tissue is a unique asset that will provide quality assurance across the Program Project.