Developing a model for the study of respiratory inflammation in the zebrafish

开发斑马鱼呼吸道炎症研究模型

• 批准号: NC/M001512/1
• 负责人: Margaret Dallman
• 金额: $ 12.54万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FM001512%2F1
• 关键词: Developing; model; study; respiratory; inflammation

Severe asthma is a disabling non-curable condition that can only be controlled by continuous administration of drugs. It has long been suggested that patients with a history of viral infection together with smoking have increased chances of developing severe asthma. Unfortunately, the pathophysiological mechanisms of this disease are not fully understood. More research is needed to shed light on how asthma develops in some people and to provide new targets for the pharmaceutical industry.We propose to investigate whether and to what extent we could use zebrafish as a model to help us understand some of the basic mechanisms and pathway biology of severe asthma in humans. Although zebrafish do not have lungs, their gills serve the same function and have the same overall tissue structure and cell types as human lungs: an epithelial cell layer that facilitates gaseous exchange and a variety of immune cells that defend the tissue from infection. A major advantage is that gills are directly exposed to ambient water, which makes them easily accessible to drugs (addition to the water rather than by injection) or for collection of biopsies. Further, we can easily access the gill tissue under brief anaesthesia to take small biopsies instead of using invasive procedures like bronchoalveolar lavage as performed in mouse studies, where a small tube is inserted through the mouth into the lungs and fluid is injected and retrieved for analysis. In fact, we can take small biopsies a number of times from the same animal in longitudinal studies, reducing the number of animals otherwise used and providing us with higher quality data by avoiding the inherent variability that exists between individuals. In addition, we have a number of fish in which specific immune cells are labeled with fluorescence. Using microscopy we will visualise, in live fish, the behaviour of these cells under different conditions including exposure to smoke and/or virus which will enable study of the same animal over time, again reducing the total number of animals used.This project will be performed in collaboration with our clinical colleagues at St Mary's Hospital who have already developed a platform which allows analysis of nasal samples in human volunteers (smokers and non-smokers) to study the effect of viral infection combined with smoking on lung function. Their experience will be invaluable in helping us develop non-invasive sampling in zebrafish and to allow the comparison of results across the species to work towards the identification of new therapeutic targets for respiratory disease. We will then be in a position to directly test the effects of intervention in these putative pathways and/or molecules on gill function in the fish.Knowledge of fish husbandry, biology and behavior are essential for this project. Therefore, the work on zebrafish will be performed by one of our most experienced scientists who has been working in the field of zebrafish immunology for 5 years and who has already developed a number of the experimental procedures required for this proposal. This will minimise the time needed for training.During this year-long project we aim to first develop methods for non-invasive sampling of gill fluids and tissues. We will apply these methods more than once on the same animal allowing studies over time on the same animal to obtain high quality data using fewer animals. During the second part of the project we will analyse our experimental samples and study many events from when, where and what type of cells are recruited to which genes/pathways play a role in inflammation. The results collected will eventually be compared with those obtained in the parallel study in humans to enable us to identify molecules or cells that play a critical role in the initiation of severe asthma. We hope to obtain a better understanding of the mechanisms of severe asthma in order to eventually develop successful treatments for this recalcitrant disease.

严重哮喘是一种无法治愈的致残性疾病，只能通过持续服药来控制。长期以来，人们一直认为，有病毒感染史和吸烟的患者患严重哮喘的机会增加。不幸的是，这种疾病的病理生理机制还不完全清楚。需要更多的研究来阐明哮喘是如何在某些人中发生的，并为制药行业提供新的靶点。我们建议调查是否以及在多大程度上可以使用斑马鱼作为模型，以帮助我们了解人类严重哮喘的一些基本机制和途径生物学。虽然斑马鱼没有肺，但它们的鳃具有与人类肺相同的功能和整体组织结构和细胞类型：促进气体交换的上皮细胞层和保护组织免受感染的各种免疫细胞。一个主要的优点是，鳃直接暴露在周围的水中，这使得它们很容易被药物接触(除了水之外，而不是通过注射)或用于收集活组织检查。此外，我们可以在短暂的麻醉下很容易地接触到鳃组织，进行小范围的活组织检查，而不是像小鼠研究中那样使用侵入性程序，如支气管肺泡灌洗，即通过口腔将一条小管插入肺部，并注射和回收液体进行分析。事实上，在纵向研究中，我们可以多次从同一动物身上进行小活检，减少了其他用途的动物的数量，并通过避免个体之间存在的内在差异，为我们提供了更高质量的数据。此外，我们还有一些鱼的特定免疫细胞是用荧光标记的。利用显微镜，我们将在活鱼身上显示这些细胞在不同条件下的行为，包括暴露在烟雾和/或病毒中，这将使随着时间的推移对同一动物的研究成为可能，再次减少使用的动物总数。该项目将与我们在圣玛丽医院的临床同事合作进行，他们已经开发了一个平台，可以分析人类志愿者(吸烟者和不吸烟者)的鼻样，以研究病毒感染和吸烟对肺功能的影响。他们的经验将有助于我们开发斑马鱼的非侵入性采样，并允许比较不同物种的结果，以确定呼吸系统疾病的新治疗靶点。然后，我们将能够直接测试干预这些假定的途径和/或分子对鱼类鳃功能的影响。有关鱼类养殖、生物学和行为的知识对这个项目至关重要。因此，关于斑马鱼的工作将由我们在斑马鱼免疫学领域工作了5年的最有经验的科学家之一进行，他已经开发了这项提议所需的一些实验程序。这将最大限度地减少培训所需的时间。在这个长达一年的项目中，我们的目标是首先开发非侵入性取样方法，对腮液和组织进行采样。我们将不止一次地在同一动物身上应用这些方法，以便随着时间的推移，在同一动物上进行研究，以使用更少的动物来获得高质量的数据。在项目的第二部分，我们将分析我们的实验样本，并研究从何时、何地、什么类型的细胞招募到哪些基因/途径在炎症中发挥作用的许多事件。收集的结果最终将与在人类身上进行的平行研究中获得的结果进行比较，以使我们能够识别在严重哮喘的发病过程中发挥关键作用的分子或细胞。我们希望更好地了解重症哮喘的发病机制，以便最终开发出治疗这种顽固性疾病的成功方法。