Proteomic Profile of Brain Endothelial Cell in Hypoxia

缺氧状态下脑内皮细胞的蛋白质组学特征

• 批准号: 6959803
• 负责人: MINGMING NING
• 金额: $ 20.23万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-05 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6959803
• 关键词: affinity labeling; bioinformatics; biotechnology; brain cell; cell cell interaction; cellular pathology; cerebral ischemia /hypoxia; clinical research; cytoplasm; electrospray ionization mass spectrometry; endopeptidases; human tissue; immunocytochemistry; metalloendopeptidases; microarray technology; oxidative stress; posttranslational modifications; proteomics; reperfusion; stroke; tissue /cell culture; vascular endothelium; western blottings

DESCRIPTION (provided by applicant): The emerging concept of the neurovascular unit emphasizes that cell-cell interactions between vascular and parenchymal compartments mediate an integrative response to injury. Thus, endothelial cell dysfunction may be an upstream trigger for brain damage after stroke. For this R21 Project, we propose to investigate the Proteomic Profiles of Human Brain Endothelial Cells in Hypoxia, to broadly characterize the role of soluble and cytosolic mediators in this in vitro stroke model. Building on our encouraging preliminary findings of novel proteinases and substrates found in oxidative stress, we hypothesize that: In comparison to controls, human endothelial cell cultures exposed to hypoxic insult will have consistent and identifiable soluble-protein and substrate expression profiles, indicating relative up- or down-regulation of both known and unknown factors important in cell-cell and cell-matrix signaling. We propose a three-part study in human brain endothelial cell culture media and lysates, to investigate the mechanism of hypoxia-reoxygenation. Specific Aim 1: Using validated techniques of quantitative proteomics technology, examine the protein expression profile of human endothelial cell culture media pre and post hypoxia-reoxygenation. Specific Aim 2: Examine the down-stream substrates repertoire of human endothelial cell culture media pre and post hypoxia-reoxygenation, in order to obtain relative quantification of the substrate repertoire characteristic of hypoxic injury. Secondary Aims: Extend Aim 1 and 2 to study cell lysates. Through quantitative proteomic analysis of hypoxia-reoxygenation in human brain endothelial cells, an important component of the neurovascular unit, we hope to provide a screening tool for the discovery of novel proteins in ischemic models, and provide venues for the discovery of new therapeutic targets.

描述（由申请人提供）：神经血管单位的新兴概念强调血管和实质隔室之间的细胞-细胞相互作用介导对损伤的综合反应。因此，内皮细胞功能障碍可能是中风后脑损伤的上游触发因素。对于这个R21项目，我们建议研究缺氧条件下人脑内皮细胞的蛋白质组学谱，以广泛表征可溶性和胞质介质在该体外中风模型中的作用。建立在我们令人鼓舞的初步发现的新的蛋白酶和底物中发现的氧化应激，我们假设：在对照组相比，暴露于缺氧损伤的人内皮细胞培养物将具有一致的和可识别的可溶性蛋白质和底物的表达谱，表明相对上调或下调的细胞-细胞和细胞-基质信号的重要的已知和未知的因素。我们提出了一个三部分的研究在人脑内皮细胞培养基和裂解物，探讨缺氧-复氧的机制。具体目标1：采用定量蛋白质组学技术，检测缺氧-复氧前后人内皮细胞培养液中蛋白质的表达谱。具体目标二：检查缺氧-复氧前后人内皮细胞培养基的下游底物库，以获得缺氧损伤特征性底物库的相对定量。次要目的：扩展目的1和2以研究细胞裂解物。通过对人脑内皮细胞缺氧-复氧的定量蛋白质组学分析，我们希望为缺血模型中发现新的蛋白质提供筛选工具，并为发现新的治疗靶点提供场所。