Pre-Clinical Models of VILI /ARDS Core

摘要： CORE C，呼吸机相关性肺损伤(VILI)/急性呼吸窘迫综合征的临床前模型 (ARDS)，旨在为PPG调查人员提供定义严格且可重现的小鼠模型 VILI，一种同时暴露于呼吸机和脂多糖(LPS)所致的二次打击肺损伤模型 更好地模拟患者ARDS的给药(VILI+LPS)，以及大肠杆菌肺炎模型。核心C将 通过以下方式全面生成、管理和提供所有与动物相关的实验、资源和专业知识 完成6个具体目标。第一个具体目标将提供全面的专业知识、培训、 设备和数据分析工具，以广泛研究临床前模型的致病机制 小鼠肺损伤。我们将使用最先进的技术来a)表征各种细胞内的角色 调节肺内皮细胞(EC)屏障功能的信号级联反应，b)决定特异性 干预措施，以深入了解新的治疗策略的疗效和机制；以及c) 促进将基础研究转化为临床干预措施。为了实现这些目标，Core C将首先提供 验证了血管通透性和炎症的定量测量。具体目标#2将包含 并照顾基因工程小鼠并产生新的转基因和基因敲除小鼠(例如， 可诱导内皮细胞特异性和肺上皮特异性条件性基因敲除小鼠)。具体目标#3将 检测选择性siRNA或药理学药物的靶向信号级联作为潜在的治疗作用 临床前模型和最终ARDS的策略和方法。具体目标4将是提供 VILI、VILI+内毒素(“二次打击”)、大肠杆菌肺炎模型的具体实验策略 急性呼吸窘迫综合征以及针对基因的救援干预。具体目标#5将评估ARDS的功能- 翻译后功能蛋白的相关单核苷酸多态性(SNPs)和位点 修饰(PTM)，利用针对肺的突变的cDNAs(高效表达载体) 血管内皮细胞条件性基因敲除小鼠的内皮细胞(用ACE抗体偶联脂质体)。特定的 AIM#6将为数据存储和保存完好的肺组织样本提供高质量的共享数据 用于更复杂的分子和细胞分析的单个项目。核心C将集中所有与MICE相关的 在该计划的所有三个项目中开展工作，包括培育新品系、繁育和安置小鼠， 建立临床前VILI/ARDS和VILI+LPS模型，评估siRNAs和 化学物质，进行肺部炎症评估，并提供组织样本和新鲜分离的 小鼠肺血管内皮细胞对每个项目进行特定的分析(包括免疫组织化学和 免疫细胞化学、蛋白质印迹分析、荧光显微镜)。除了它自己的空间和 设备，核心C将完全访问并利用亚利桑那大学的可用资源 共享设施包括基因工程小鼠模型核心(GEMM)。

SUMMARY: Core C, Pre-Clinical Models of ventilator-induced lung injury (VILI)/acute respiratory distress syndrome (ARDS), is designed to provide PPG investigators with rigorously defined and reproducible murine models of VILI, a two-hit lung injury model induced by exposure to both a ventilator and lipopolysaccharide (LPS) administration (VILI+LPS) that better mimics ARDS in patients, and an E. coli pneumonia model. Core C will comprehensively generate, manage and provide all animal-related experiments, resources, and expertise by accomplishing 6 specific aims. Specific Aim #1 will provide a complete range of expertise, training, equipment, and data analysis tools to extensively study the pathogenic mechanisms in preclinical models of murine lung injury. We will employ state-of-the-art techniques to a) characterize the role of various intracellular signaling cascades in regulating lung endothelial cell (EC) barrier function, b) determine the effects of specific interventions to provide insight into the efficacy and mechanisms of novel therapeutic strategies; and c) facilitate the translation of basic research to clinical interventions. Toward these goals, Core C will first provide validated quantitative measurements of vascular permeability and inflammation. Specific Aim #2 will house and care for the genetically-engineered mice and to generate novel transgenic and knockout mice (e.g., inducible endothelium-specific and lung epithelium-specific conditional knockout mice). Specific Aim #3 will examine selective siRNAs or pharmacological agents for target signaling cascades as potential therapeutic strategies and approaches for preclinical models and ultimately, for ARDS. Specific Aim #4 will be to provide performance of specific experimental strategies involving VILI, VILI+LPS (“two-hit”), E. coli pneumonia models of ARDS as well as gene-specific rescue interventions. Specific Aim #5 will evaluate the function of ARDS- associated single nucleotide polymorphisms (SNPs) and sites of functional protein post-translational modification (PTM), utilizing mutated cDNA (high efficiency expression plasmids) targeting the lung endothelium (with ACE antibody-conjugated liposome) in the endothelial conditional knockout mice. Specific Aim #6 will provide high quality shared data for data storage and well-preserved lung tissue samples to individual projects for more sophisticated molecular and cellular assays. Core C will centralize all mice-related work across all three projects of this program, including generating new strains, breeding and housing of mice, generating preclinical VILI/ARDS and VILI+LPS models, accessing therapeutic effects of siRNAs and chemicals, performing lung inflammation assessment, and providing tissue samples and freshly dissociated murine lung vascular endothelial cells to each project for specific assays (including immunohisto- and immunocytochemistry, western blot analysis, fluorescent microscopy). In addition to its own space and equipment, Core C will have full access to and will utilize resources available at the University of Arizona shared facilities including the Genetically Engineered Mouse Models Core (GEMM).