Thioredoxin in Neutrophil-Airway Epithelial Interactions

描述（由申请人提供）： 本研究的主要目的是确定氧化损伤后硫氧还蛋白在嗜中性粒细胞-气道上皮细胞相互作用中的重要性。 中性粒细胞通过清除氧化剂损伤的气道上皮细胞和增强环境臭氧暴露后随后的上皮修复发挥有益作用。中心假设是嗜中性粒细胞依赖性去除氧化剂损伤的气道上皮细胞需要由损伤细胞产生硫氧还蛋白，然后增强嗜中性粒细胞共定位，去除损伤细胞和随后的上皮修复。本提案的目标将分四个部分阐述。 首先，研究人员将确定硫氧还蛋白和氧化损伤的气道上皮细胞的细胞膜之间的分子相互作用。 其次，他们将研究硫氧还蛋白在中性粒细胞与气道上皮细胞共定位、损伤后清除和增强后续上皮修复中的重要性。 永生化的人支气管上皮细胞将用硫氧还蛋白表达构建体稳定转染，研究人员将定量中性粒细胞与这些细胞的共定位，嗜中性粒细胞依赖性去除氧化损伤的细胞和使用BrdU的上皮修复。为了评估硫氧还蛋白结构在这些功能中的重要性，研究人员将使用在关键半胱氨酸氨基酸中突变的硫氧还蛋白表达构建体转染的单层细胞重复实验。第三，他们将证明硫氧还蛋白对小鼠中性粒细胞具有趋化性。这将导致第四个目的，即开发在气道上皮中过表达野生型和突变型硫氧还蛋白的转基因小鼠，并证明硫氧还蛋白在体内中性粒细胞迁移和去除损伤细胞中的作用。主要候选人的长期研究目标是研究肺部炎症和修复的机制，特别强调中性粒细胞和气道上皮细胞之间的相互作用。 她希望利用职业研究奖来获得分子生物学方面的额外知识和培训，同时过渡到一个独立的研究人员谁将有竞争力的R 01资金。

DESCRIPTION (provided by applicant): The primary objective of this research is to determine the importance of thioredoxin in neutrophil-airway epithelial cell interactions after oxidant injury. Neutrophils play a beneficial role by removing oxidant-injured airway epithelial cells and enhancing subsequent epithelial repair after ambient ozone exposure. The central hypothesis is that neutrophil-dependent removal of oxidant-injured airway epithelial cells requires thioredoxin production by the injured cells that then enhances neutrophil co-localization, removal of injured cells and subsequent epithelial repair. The objectives of this proposal will be addressed in four parts. First, the investigators will determine the molecular interaction between thioredoxin and the cell membrane of oxidant-injured airway epithelial cells. Secondly, they will investigate the importance of thioredoxin in neutrophil co-localization to airway epithelial cells, their removal after injury and enhancing subsequent epithelial repair. Immortalized human bronchial epithelial cells will be stably transfected with a thioredoxin expression construct and the investigators will quantify neutrophil co-localization to these cells, neutrophil-dependent removal of oxidant injured cells and epithelial repair using BrdU. To assess the importance of thioredoxin structure in these functions, the investigators will repeat the experiments using monolayers transfected with thioredoxin expression constructs mutated in key cysteine amino acids. Third, they will show thioredoxin is chemotactic for mouse neutrophils. This will lead to the fourth aim of developing a transgenic mouse over-expressing wild type and mutant thioredoxin in airway epithelium and demonstrating the role of thioredoxin in neutrophil emigration and removal of injured cells in vivo. The primary candidate's long-term research goals are to investigate mechanisms of pulmonary inflammation and repair with special emphasis on the interaction between neutrophils and airway epithelial cells. She hopes to use a career research award to gain additional knowledge and training in molecular biology while transitioning to an independent researcher who will be competitive for R01 funding.