Monitoring cellular redox signaling and oxidant stress in vivo

监测体内细胞氧化还原信号和氧化应激

• 批准号: 7918913
• 负责人: PAUL T SCHUMACKER
• 金额: $ 24.7万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7918913
• 关键词: Adenoviruses; Affect; Age; Animal Disease Models; Animals; Awareness; Behavior; Biological Models; Breathing; Breeding; Cardiovascular system; Cell Communication; Cell Culture Techniques; Cell physiology; Cells; Cysteine; Cytosol; DNA; Data; Diabetes Mellitus; Disease; Disease model; Electron Transport; Electron Transport Complex III; Environmental air flow; Event; Fluorescence; Fluorescence Microscopy; Functional disorder; Gases; Generations; Genes; Genomics; Health; Hypoxia; Induced Mutation; Inflammation; Kidney Diseases; Knock-in Mouse; Lead; Learning; Life; Lipids; Liver diseases; Lung; Malignant Neoplasms; Measures; Membrane; Methods; Microscopy; Mitochondria; Mitochondrial Matrix; Monitor; Mus; Myocardial Ischemia; Neurodegenerative Disorders; Organ; Outcome; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxygen; Parkinson Disease; Pathogenesis; Photons; Physiological; Primary Cell Cultures; Process; Proteins; Pulmonary artery structure; Reaction; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reporter; Reporter Genes; Research; Research Personnel; Role; Second Messenger Systems; Signal Transduction; Signal Transduction Pathway; Site; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Stress; Stroke; Sulfhydryl Compounds; System; Technology; Terminator Codon; Testing; Tissue Harvesting; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Vascular Diseases; base; cell injury; cell type; free radical oxygen; homologous recombination; in vivo; interest; mouse Cre recombinase; novel; oxidant stress; oxidative damage; performance tests; public health relevance; recombinase; response; second messenger; sensor; tool; two-photon

DESCRIPTION (provided by applicant): Healthy cells use low levels of reactive oxygen species (ROS) as second messengers in signal transduction pathways. High levels of ROS cause oxidative damage to proteins, lipids and DNA. Oxidant stress has been implicated in the cellular dysfunction associated with ischemia- reperfusion injury, vascular disease, stroke, diabetes, neurodegenerative diseases, liver disease, renal disease, inflammation, cancer, and other disorders. As the awareness of the role of redox stress in health and disease has grown, the demand for new tools to monitor oxidant stress in vivo has increased. Current methods to assess redox events are limited by their inability to provide quantitative data or spatial information on the subcellular sites of oxidant generation. Moreover, existing probes are generally unsuitable for in vivo studies. New methods to monitor intracellular oxidant stress in intact tissues could enhance our understanding of how cell-cell interactions and tissue microenvironments influence the generation of ROS. We propose to create a new system to detect redox status and oxidative stress in specific cells within intact tissues, using a novel combination of existing methods. In Aim 1 we will create transgenic mice with DNA encoding the redox-sensitive fluorescent protein, RoGFP, inserted at a LoxP-silenced ROSA26 genomic locus. Three lines will be generated, which target the RoGFP sensor to cytosol, mitochondrial matrix, or mitochondrial intermembrane space. In Aim 2 we will activate expression of the RoGFP genes in primary cells cultured from these mice, using Cre recombinase to delete the stop codon. We will confirm correct targeting of the expressed protein, and confirm its function in response to redox stress. In Aim 3 we will breed the RoGFP mice with smooth muscle-specific Cre recombinase mice, to elicit RoGFP expression in pulmonary artery smooth muscle cells in the lung. Using that model system to demonstrate efficacy, we will measure redox changes in smooth muscle cells in the intact lung during ventilation with different concentrations of oxygen. Two-photon microscopy will be used to assess the redox status of the subcellular targeted RoGFP proteins in vivo. These animals will therefore provide exciting new tools that will enable us, and other investigators, to monitor subcellular oxidative stress in intact tissue in diverse cell types and disease models. Public Health Relevance Statement: Healthy cells in the body use oxygen free radicals (Reactive Oxygen Species, or ROS) to regulate various cellular functions. Excessive levels of ROS disrupt cell function, and they contribute to cellular injury in a large number of diseases. To understand how ROS affect cells, it is essential to monitor their levels. However, current tools are limited in their ability to assess intracellular ROS. We propose to correct this problem by inserting a gene encoding an ROS-sensitive fluorescent protein into mice. When the gene is turned on, the cell will generate a protein that moves to a known intracellular compartment and signals a change in ROS levels by altering its fluorescence. We will turn this gene on in certain types of cells in the mouse, and measure the fluorescence changes using a form of microscopy that can "see" deeply into intact tissues. We will test the performance of this sensor in the lungs, where we will measure the ROS response to changes in the concentration of oxygen that the animal is breathing. However, many other investigators will be able to use the same mice where, by turning on the reporter gene in other cell types, it will be possible to monitor ROS in a wide range of different tissues. Hence, this mouse will provide useful information on ROS levels in a wide range of disease models. The successful outcome of this project is supported by extensive preliminary studies demonstrating the feasibility of each step in the process. The end result should significantly extend our ability to assess ROS in intact tissues, in animal models of disease.

描述（由申请人提供）：健康细胞使用低水平的活性氧（ROS）作为信号转导途径中的第二信使。高水平的ROS会对蛋白质、脂质和DNA造成氧化损伤。氧化应激已经涉及与缺血-再灌注损伤、血管疾病、中风、糖尿病、神经变性疾病、肝病、肾病、炎症、癌症和其它病症相关的细胞功能障碍。随着人们对氧化还原应激在健康和疾病中的作用的认识不断提高，对监测体内氧化应激的新工具的需求也在增加。目前的方法来评估氧化还原事件是有限的，他们无法提供定量数据或空间信息的亚细胞网站的氧化剂生成。此外，现有的探针通常不适合于体内研究。监测完整组织中细胞内氧化应激的新方法可以增强我们对细胞间相互作用和组织微环境如何影响ROS产生的理解。我们建议创建一个新的系统来检测完整组织内特定细胞的氧化还原状态和氧化应激，使用现有方法的新组合。在目标1中，我们将创建具有编码氧化还原敏感性荧光蛋白RoGFP的DNA的转基因小鼠，所述DNA插入在LoxP沉默的ROSA 26基因组位点。将产生三条线，其将RoGFP传感器靶向细胞质、线粒体基质或线粒体膜间隙。在目标2中，我们将使用Cre重组酶删除终止密码子，激活从这些小鼠培养的原代细胞中RoGFP基因的表达。我们将确认表达蛋白的正确靶向，并确认其响应氧化还原应激的功能。在目标3中，我们将用平滑肌特异性Cre重组酶小鼠繁殖RoGFP小鼠，以引起RoGFP在肺动脉平滑肌细胞中的表达。使用该模型系统来证明疗效，我们将测量在不同浓度的氧气通气期间完整肺中平滑肌细胞的氧化还原变化。双光子显微镜将用于评估体内亚细胞靶向RoGFP蛋白的氧化还原状态。因此，这些动物将提供令人兴奋的新工具，使我们和其他研究人员能够监测不同细胞类型和疾病模型中完整组织中的亚细胞氧化应激。 公共卫生相关性声明：体内的健康细胞使用氧自由基（活性氧，或ROS）来调节各种细胞功能。ROS水平过高会破坏细胞功能，并导致大量疾病中的细胞损伤。为了了解ROS如何影响细胞，监测其水平至关重要。然而，目前的工具在评估细胞内ROS的能力方面是有限的。我们建议通过将编码ROS敏感荧光蛋白的基因插入小鼠来纠正这个问题。当基因被打开时，细胞将产生一种蛋白质，该蛋白质移动到已知的细胞内区室，并通过改变其荧光来发出ROS水平变化的信号。我们将在小鼠的某些类型的细胞中开启这种基因，并使用一种可以“看到”完整组织深处的显微镜来测量荧光变化。我们将在肺部测试这种传感器的性能，在那里我们将测量ROS对动物呼吸的氧气浓度变化的反应。然而，许多其他研究人员将能够使用相同的小鼠，通过在其他细胞类型中打开报告基因，将有可能在广泛的不同组织中监测ROS。因此，这种小鼠将在广泛的疾病模型中提供有关ROS水平的有用信息。该项目的成功结果得到了广泛的初步研究的支持，这些研究证明了该过程中每个步骤的可行性。最终的结果应该显着扩展我们的能力，以评估活性氧在完整的组织，在动物模型的疾病。