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

暴露于污染物的人气道细胞中 EGFR 活性的单细胞分析

• 批准号: 8391214
• 负责人: Ryan Matthew Phillips
• 金额: $ 3.28万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391214
• 关键词: Affect; Air; Air Pollution; Area; Automobiles; Biopsy Specimen; Blood Pressure; Breathing; Bronchoalveolar Lavage; Capillary Electrophoresis; Cardiac; Cardiovascular Diseases; Cause of Death; Cell Surface Proteins; Cells; Cessation of life; Charge; Communicable Diseases; Cytolysis; Deposition; Detection; Development; Diesel Exhaust; Diesel Fuels; Diet; Ensure; Environment; Enzymes; Epidemiologic Studies; Epidemiology; Epidermal Growth Factor Receptor; Epithelial Cells; Event; Exclusion; Fluorescence; Foreign Bodies; Generations; Genetic; Glass; Health; Heart Diseases; Human; IL8 gene; Individual; Inflammatory Response; JNK-activating protein kinase; Label; Lasers; Lead; Libraries; Life; Link; Lung Inflammation; Lung diseases; MAPK14 gene; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Methods; Microscope; Mitogen-Activated Protein Kinases; Myocardial Infarction; Particulate Matter; Peptide Hydrolases; Peptides; Performance; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Play; Pollution; Population; Process; Protein Fragment; Protein Tyrosine Phosphatase; Proteins; Public Health; Reaction; Reactive Oxygen Species; Receptor Activation; Recycling; Regulation; Reporter; Research; Risk; Role; Safety; Sample Size; Sampling; Secondary to; Signal Transduction; Specimen; Stimulus; Stroke; Techniques; Technology; Testing; Time; Transcription Factor AP-1; Ultrafine; Western Blotting; Woman; Work; air pollution control; airway epithelium; airway inflammation; base; cell growth; environmental agent; established cell line; heart rhythm; human subject; in vivo; inorganic phosphate; interest; meetings; men; nanosecond; new technology; particle; particle exposure; peptide structure; pollutant; public health relevance; response; single cell analysis; upstream kinase

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.

描述（由申请人提供）：心血管疾病是美国男性和女性死亡的主要原因，2006年导致34%的死亡。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不完全清楚，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。虽然人们认为EGFR在因污染物吸入引起的肺部炎症中起着重要作用，但在活体人体气道样本中直接测量EGFR活性目前尚不可行。这项研究将开发一种能够测量EGFR活性的新技术，从而可以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。在这项提案中开发的技术包括将一个小的荧光蛋白片段或肽放入活细胞中，允许它通过添加磷酸基团（磷酸化）被EGFR修饰，然后使用毛细管电泳测量这一过程的速率，毛细管电泳允许根据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废气颗粒的细胞中的EGFR活性，首先使用在实验室中培养的细胞，然后使用直接从人体受试者身上取下的活检标本，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，使其非常适合这种应用。为了有效地测量细胞中的EGFR活性，肽需要被EGFR有效地磷酸化，而不是被相关酶磷酸化。此外，它必须能够抵抗细胞内被称为蛋白酶的酶的降解，蛋白酶可以分解蛋白质进行循环利用并消除异物。为了确保满足这些要求，将使用已建立的基于文库的方法修改肽的结构。该项目将巩固空气污染与导致心脏病的肺部炎症之间的联系，并为支持最有利于人类健康的环境法规提供强有力的证据。此外，这项技术一旦开发出来，也将很容易适用于其他研究领域，包括癌症和传染病。