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

描述（由申请人提供）：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物会存款在气道中，导致血压升高、心律改变，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机制尚未完全了解，但气道炎症似乎起着重要作用。 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部刺激的反应，包括炎症反应。虽然认为EGFR在由于污染物吸入引起的肺部炎症中起重要作用，但目前在活体人气道样品中直接测量EGFR活性是不可行的。这项研究将开发一种能够测量EGFR活性的新技术，从而可以分析这些样本，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。 该提案中开发的技术涉及将小的荧光蛋白片段或肽放入活细胞中，通过添加磷酸基团（磷酸化）使其被EGFR修饰，然后使用毛细管电泳测量该过程的速率，该毛细管电泳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于柴油机废气颗粒的细胞中的EGFR活性，首先使用实验室中生长的细胞，然后直接从人类受试者中取出活检标本，由于可以获得的活细胞数量很少，这在以前是不可行的。所提出的技术能够分析单细胞，使其非常适合这种应用。 为了有效地测量细胞中的EGFR活性，肽将需要被EGFR而不是相关酶有效地磷酸化。此外，它必须能够抵抗细胞内称为蛋白酶的酶的降解，蛋白酶分解蛋白质以进行回收并消除异物。为确保满足这些要求，将使用已建立的基于文库的方法修饰肽的结构。 该项目将巩固空气污染与肺部炎症导致心脏病之间的联系，并提供强有力的证据支持符合人类健康最佳利益的环境法规。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。 公共卫生相关性：这项工作将探索柴油废气颗粒（空气污染的常见成分）导致气道炎症并导致肺部和心脏疾病的机制。了解这一过程的细节将有助于通过指导控制空气污染的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.