Monitoring cellular redox signaling and oxidant stress in vivo

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。高水平的活性氧会对蛋白质、脂质和DNA造成氧化损伤。氧化应激与缺血-再灌注损伤、血管疾病、中风、糖尿病、神经退行性疾病、肝脏疾病、肾脏疾病、炎症、癌症和其他疾病相关的细胞功能障碍有关。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，对体内氧化应激监测新工具的需求也在增加。目前评估氧化还原事件的方法由于无法提供氧化生成亚细胞位点的定量数据或空间信息而受到限制。此外，现有的探针通常不适合体内研究。在完整组织中监测细胞内氧化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生成的理解。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Aim 1中，我们将创建转基因小鼠，将编码氧化还原敏感荧光蛋白RoGFP的DNA插入loxp沉默的ROSA26基因组位点。将产生三条线，将RoGFP传感器定位于细胞质、线粒体基质或线粒体膜间空间。在目标2中，我们将在这些小鼠培养的原代细胞中激活RoGFP基因的表达，使用Cre重组酶删除停止密码子。我们将确认表达蛋白的正确靶向性，并确认其在氧化还原应激反应中的功能。在Aim 3中，我们将RoGFP小鼠与平滑肌特异性Cre重组酶小鼠杂交，以诱导肺动脉平滑肌细胞中RoGFP的表达。使用该模型系统来证明有效性，我们将测量不同浓度氧气通气时完整肺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在不同细胞类型和疾病模型的完整组织中监测亚细胞氧化应激。

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.