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离体人类平台，以研究机制 调节人气道平滑肌（HASM）中的兴奋-收缩（EC）偶联。核心B的目标 建立、表征和提供原代HASM细胞，生成 细胞骨架（CSK）刚度和力产生（磁扭转细胞术- MTC），并进行人 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 在原代HASM细胞的建立、培养和表征中。核心B也将提供哮喘- 和非哮喘衍生的HASM，以表征疾病状态产生的内在差异， 人类反应的异质性。hPCLS将作为测量生理相关性的平台 在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。目的1为HASM细胞提供新的细胞模型 高反应性，以探讨EC偶联，单细胞缩短和力之间的关系 GPCR激动剂和拮抗剂在哮喘中的作用。利用这些模型，项目1将研究 TGF-β1调节气道收缩的潜在机制;项目2和4将阐明机制 调节TAS 2 R和OGR 1介导的HASM松弛;项目3将开发靶向治疗药物， 减弱HASM的收缩性并增强其松弛性。目的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。使用hPCLS，项目 1将研究TGF-β1调节小气道收缩和扩张的机制;项目2和 项目4将阐明TAS 2 R和ORG 1介导小气道扩张的机制;项目3将 发现新的方法来消除AHR和增强靶向治疗的支气管扩张。目标3将 阐明炎症介质改变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TAS 2 R和OGR 1介导的支气管扩张在收缩性中的作用。 在细胞因子暴露或肥大细胞脱粒的背景下;项目3将开发配体， AHR和恢复支气管扩张剂低反应性，尽管T2炎症。核心B的贡献是 重要的是，我们的模型将阐明消除AHR和增强支气管扩张的分子靶点， 哮喘的背景。核心B的创新点在于最先进的单细胞力测量 使用体外和离体模型研究HASM的产生和小气道功能。此外，我们的模型仅使用 人类细胞和组织减轻物种差异，混淆EC耦合ASM的研究。一 集中的人类细胞和组织核心擅长研究HASM细胞和组织是一个独特的资产， 为整个项目提供质量保证。

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.