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

基本信息

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
项目类别：
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

项目摘要

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女死亡的主要原因，在2006年导致34％的死亡。研究表明，诸如柴油燃料燃烧（例如柴油燃料燃烧）释放到空气中的小颗粒可以沉积在呼吸道中，并导致血压的增加，心脏病的变化，心脏病的风险增加，并增加了心脏病风险，并增加了心脏病的风险。尽管尚未完全了解这些健康影响的机制，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由于吸入污染物引起的肺部炎症起着重要作用，但目前不可行地测量Live人类气道样品中EGFR活性的直接测量。这项研究将开发一种能够测量EGFR活动的新技术，该技术将允许对此类样本进行分析，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。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，从而可以通过添加磷酸盐组（磷酸化）将其通过EGFR对其进行修改，然后使用毛细血管电泳进行测量该过程的速率，从而使分子基于差异的分离和测量，以差异和计算。该项目的目的是比较健康细胞中的EGFR活性和暴露于柴油排气颗粒的活性，首先使用实验室中生长的细胞，然后在直接从人类受试者中取出的活检标本中，这是由于可以获得的活细胞数量少而不可行的。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，需要通过EGFR有效地磷酸化肽，但没有相关的酶。同样，它必须能够抵抗称为蛋白酶的细胞中的酶降解，该酶分解蛋白质以回收并消除异物。为了确保满足这些要求，将使用建立的基于库的方法对肽的结构进行修改。该项目将巩固空气污染与肺部炎症之间的联系，导致心脏病，并提供有力的证据，以支持符合人类健康最大利益的环境法规。此外，一旦开发，该技术也将很容易适应其他研究领域，包括癌症和传染病。