Single Cell Analysis of EGFR Activity in Human Airway Cells Exposed to Pollutants

简介(申请人提供)：心血管疾病是美国男性和女性的主要死亡原因，2006年占死亡人数的34%。研究表明，柴油燃烧等过程释放到空气中的小颗粒会沉积在呼吸道中，导致血压升高、心率改变，并显著增加心脏病发作和中风的风险。虽然这些健康影响的潜在机制尚不完全清楚，但呼吸道炎症似乎发挥了重要作用。表皮生长因子受体(EGFR)是一种细胞表面蛋白，有助于调节细胞生长和对外界刺激(包括炎症反应)的反应。虽然人们认为EGFR在污染物吸入引起的肺部炎症中起着重要作用，但目前还不能直接测量活体人类呼吸道样本中的EGFR活性。这项研究将开发一种能够测量EGFR活性的新技术，从而能够对这些样本进行分析，并将提供有关空气污染、肺部炎症和心脏病之间联系的重要信息。这项提案中开发的技术涉及将一个小的荧光蛋白片段或肽放入活细胞中，允许EGFR通过添加磷酸基团(磷酸化)对其进行修饰，然后使用毛细管电泳法测量这一过程的速度，毛细管电泳法允许基于大小和电荷的差异分离和测量分子。该项目的目的是比较健康细胞和暴露在柴油废气颗粒物中的EGFR活性，首先使用在实验室培养的细胞，然后从直接从受试者身上取出的活组织标本中进行比较，这在以前是不可行的，因为可以获得的活细胞数量很少。所提出的技术能够分析单个细胞，使其非常适合于这样的应用。为了有效地测量细胞中的EGFR活性，多肽需要被EGFR有效地磷酸化，而不是相关的酶。此外，它还必须能够抵抗细胞内被称为蛋白酶的酶的降解，这种酶能分解蛋白质以供循环使用，并清除异物。为了确保满足这些要求，将使用已建立的基于文库的方法来修改多肽的结构。该项目将巩固空气污染和导致心脏病的肺部炎症之间的联系，并为支持符合人类健康最佳利益的环境法规提供强有力的证据。此外，一旦开发出来，这项技术也将很容易适应其他研究领域，包括癌症和传染病。 与公共健康相关：这项工作将探索柴油废气颗粒物--空气污染的常见成分--导致呼吸道炎症并导致肺病和心脏病的机制。了解这一过程的细节将有助于通过指导空气污染控制法规来保护人类健康和安全。

DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of death in the US for both men and women, causing 34% of deaths in 2006. Studies have shown that the small particles released into the air by processes such as diesel fuel combustion can deposit in the airways and lead to increases in blood pressure, changes in heart rhythm, and significantly increased risk of heart attack and stroke. While the mechanism underlying these health effects is not fully understood, airway inflammation seems to play an important role. The epidermal growth factor receptor (EGFR) is a cell surface protein that helps regulate cell growth and response to external stimuli, including the inflammatory response. While it is thought that EGFR plays an important role in lung inflammation due to pollutant inhalation, direct measurements of EGFR activity in live human airway samples are not currently feasible. This research will develop a new technology capable of measuring EGFR activity that will permit analysis of such samples, and will provide important information about the link between air pollution, lung inflammation, and heart disease. The technology developed in this proposal involves putting a small, fluorescent protein fragment, or peptide, into living cells, allowing it to be modified by EGFR by the addition of a phosphate group (phosphorylation), and then measuring the rate of this process using capillary electrophoresis, which allows the separation and measurement of molecules based on differences in size and charge. The aim of this project is to compare EGFR activity in healthy cells and those exposed to diesel exhaust particles, first using cells grown in the lab, then in biopsy specimens removed directly from human subjects, which was not previously feasible due to the small number of living cells that can be obtained. The technique proposed is capable of analyzing single cells, making it ideally suited to such an application. To effectively measure EGFR activity in cells, the peptide will need to be efficiently phosphorylated by EGFR but not related enzymes. Also, it must be able to resist degradation by enzymes within the cell known as proteases, which break down proteins for recycling and to eliminate foreign bodies. To assure these requirements are met, the structure of the peptide will be modified using an established library-based method. This project will solidify the link between air pollution and lung inflammation leading to heart disease, and provide strong evidence to support environmental regulations that are in the best interest of human health. Additionally, once developed, this technique will also be readily adapted to other areas of research, including cancer and infectious disease. PUBLIC HEALTH RELEVANCE: This work will explore the mechanism by which diesel exhaust particles, a common component of air pollution, lead to inflammation of the airways and contribute to lung and heart disease. Understanding the details of this process will help protect human health and safety by guiding regulations for controlling air pollution.