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）：健康细胞在信号转导途径中使用低水平的活性氧（ROS）作为第二使者。高水平的ROS会对蛋白质，脂质和DNA造成氧化损伤。氧化应激与与缺血再灌注损伤，血管疾病，中风，糖尿病，神经退行性疾病，肝病，肾脏疾病，炎症，炎症，癌症和其他疾病有关的细胞功能障碍有关。随着对氧化还原压力在健康和疾病中的作用的认识，对监测体内氧化剂胁迫的新工具的需求增加了。当前评估氧化还原事件的方法受到其无法在氧化剂产生的亚细胞位点提供定量数据或空间信息的限制。此外，现有探针通常不适合体内研究。监测完整组织中细胞内氧化剂应激的新方法可以增强我们对细胞 - 细胞相互作用和组织微环境如何影响ROS产生的理解。我们建议使用现有方法的新组合来创建一个新系统，以检测完整组织中特定细胞中特定细胞中特定细胞的氧化应激。在AIM 1中，我们将使用编码氧化还原敏感的荧光蛋白ROGFP的DNA创建转基因小鼠，该荧光蛋白插入了Loxp-Syleds-Rexp的Rosa26基因组基因座。将生成三条线，将ROGFP传感器靶向细胞质，线粒体基质或线粒体膜间空间。在AIM 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。我们将确认正确靶向表达的蛋白质，并确认其对氧化还原应激的功能。在AIM 3中，我们将用平滑肌特异性CRE重组酶小鼠繁殖ROGFP小鼠，以引起肺中肺动脉平滑肌细胞中的ROGFP表达。使用该模型系统来证明功效，我们将在通气过程中以不同浓度的氧气来测量完整肺中平滑肌细胞的氧化还原变化。两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各种细胞类型和疾病模型中完整组织中的亚细胞氧化应激。 公共卫生相关性陈述：体内的健康细胞使用氧气自由基（活性氧或ROS）来调节各种细胞功能。 ROS过多的细胞功能过多，它们在大量疾病中导致细胞损伤。要了解ROS如何影响细胞，必须监测其水平。但是，当前的工具在评估细胞内ROS的能力方面受到限制。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此问题。当基因打开时，该细胞将产生一种蛋白质，该蛋白质移动到已知的细胞内室，并通过改变其荧光来向ROS水平变化。我们将在小鼠中的某些类型的细胞中打开该基因，并使用一种可以深入了解完整组织的显微镜形式测量荧光变化。我们将测试该传感器在肺部的性能，在那里我们将测量动物呼吸的氧气浓度变化的ROS响应。但是，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记者基因，可以在各种不同的组织中监测ROS。因此，该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，证明了过程中每个步骤的可行性。最终结果应显着扩大我们评估疾病动物模型中完整组织中ROS的能力。