Core B - Human Cell/Tissue Acquisition and Physiology Care

项目摘要 核心B更新的主要目标是提供体外和体外人类平台来研究机制 调节人气道平滑肌(HASM)的兴奋-收缩(EC)偶联。核心B的目标 是建立、表征和提供原代HASM细胞，产生单个HASM细胞测量 细胞骨架(CSK)硬度和力产生(磁扭转细胞仪-MTC)，并执行人类 所有项目和核心的精确切割肺切片(HPCLS)研究。我们的实验室有超过25年的经验 在原代HASM细胞的建立、培养和鉴定方面。核心B还将提供哮喘- 和非哮喘衍生的HASM，以表征由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。AIM 1将提供HASM细胞的新细胞模型 高反应性探讨EC偶联、单细胞缩短和作用力之间的关系 在哮喘中产生GPCR激动剂和拮抗剂。使用这些模型，项目1将研究 转化生长因子-β-1调节呼吸道收缩的潜在机制；项目2和4将阐明机制 调节TAS2R和OGR1介导的HASM松弛；项目3将开发靶向疗法来 抑制HASM的收缩，增强其松弛。AIM 2将提供一个完整的组织模型 HASM收缩和气道高反应性(AHR)，研究GPCR信号转导。使用hPCLS，项目 1将研究转化生长因子-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调节小气道扩张的机制；项目3将 发现新的方法，以消除AHR和加强靶向治疗的支气管扩张。目标3将 阐述炎症介质改变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转化生长因子-β1对呼吸道的影响 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颗粒的背景；项目3将开发将减弱 AHR和恢复支气管扩张剂的低反应性，尽管T2炎症。核心B的贡献是 由于我们的模型将阐明消除AHR并增强支气管扩张的分子靶点，因此具有重要意义 哮喘的背景。B芯的创新集中在最先进的单胞测力测量上 用体外和体外模型研究HASM的生成和小气道功能。此外，我们的型号仅用于 人类细胞和组织减轻了对ASM中EC偶联研究的混乱的物种差异。一个 擅长研究HASM细胞和组织的集中化的人类细胞和组织核心是一项独特的资产，它将 在整个计划项目中提供质量保证。

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.