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硫氧还蛋白在氧化损伤后中性粒细胞-呼吸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损伤的呼吸道上皮细胞，并在环境臭氧暴露后加强随后的上皮修复，发挥了有益的作用。中心假说是，中性粒细胞依赖氧化剂损伤的呼吸道上皮细胞的清除需要损伤细胞产生硫氧还蛋白，然后促进中性粒细胞协同定位，清除损伤细胞和随后的上皮修复。这项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与氧化剂损伤的呼吸道上皮细胞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呼吸道上皮细胞共定位、损伤后清除中性粒细胞和促进随后的上皮修复中的重要性。永生化的人支气管上皮细胞将稳定地转染硫氧还蛋白表达载体，研究人员将量化中性粒细胞与这些细胞的共存，中性粒细胞依赖的氧化损伤细胞的清除，以及使用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，研究人员将使用硫氧还蛋白表达结构在关键半胱氨酸氨基酸中突变的单层重复实验。第三，他们将证明硫氧还蛋白对小鼠中性粒细胞具有趋化作用。这将导致第四个目标，即建立在呼吸道上皮细胞中高表达野生型和突变型硫氧还蛋白的转基因小鼠，并在体内证明硫氧还蛋白在中性粒细胞迁移和清除受损细胞中的作用。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呼吸道上皮细胞之间的相互作用。她希望利用职业研究奖获得更多的分子生物学知识和培训，同时过渡到一名将在R01资金方面具有竞争力的独立研究人员。

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.