Development of Analytical Tools for Concentration and Real-Time Control of Dissolved Gases and Their Regulation of Tissue Function

项目摘要 拟议研究的主要目标是开发工具，使溶解气体的研究， 生物样本血液气体（O2和CO2）以及痕量信号气体（NO、CO和H2S） 在调节多种组织功能如调节心率，血流， 免疫反应、激素和神经递质分泌以及细胞保护和抗炎 特性.事实上，大多数组织（如果不是全部的话）在中枢神经系统的顶部产生NO、CO和H2S并受其调节。 O2在调节生物能量学和代谢途径中的作用。学术机构的研究以及 制药公司正在努力利用气体信号的有益作用，以治疗 包括糖尿病、移植排斥、败血症、动脉粥样硬化和癌症在内的一系列疾病。尽管 溶解气体的科学和临床重要性，定量方法来测量真实的时间效应， 组织/细胞上的溶解气体不可用。研究有益信号的研究人员 由微量气体引发的和/或正在开发药物以引发相同益处的人几乎完全使用 每种信号气体的水溶性替代物/供体。该提案的一个关键点是， 气体的供体可能不允许精确控制组织内的气体水平，并且由于它们的低含水量， 溶解性，从培养基中迅速耗尽。我们生成和发布的初步数据显示， 溶解的H2S与用H2S的水性提供者获得的效果相反，表明需要重新 评估从使用供体分子的研究中建立的关于NO、H2S和CO的影响。 由于大多数生命科学研究人员没有能力研究微量气体的直接影响， 生理相关的浓度，我们将开发一个交钥匙，自动化仪器控制 组织对含有用户指定水平的6种气体的介质的浓度和暴露时间 包括NO、CO、H2S、O2、CO2和N2。这个自动化和用户友好的系统将被建造， 将气体混合物供应到各种广泛使用的组织评估模态， 平板培养和用于研究气体与纯化蛋白质结合相互作用的比色皿系统。我们有 组建了一支由具有多年经验的生物和机械/燃烧工程师组成的团队 设计和构建应用于生物分析的气体流控系统，以及应用 数学家，分析化学家和细胞生理学家，他们将参与验证 仪器仪表这项技术将对基础研究产生广泛的影响， 信号气体对组织功能、信号和生物能量学的动力学和浓度依赖性作用， 以及正在开发的模拟气体的细胞保护特性的治疗剂的评估。

Project Summary The major goal of the proposed research is to develop tools to enable the study of dissolved gases on biological samples. Both blood gases (O2, and CO2) as well as trace signaling gases (NO, CO and H2S) play critical roles in regulation of wide array of tissue functions as diverse as regulation of heart rate, blood flow, immune responses, hormone and neurotransmitter secretion, as well as cytoprotective and anti-inflammatory properties. Indeed, most if not all tissues produce and are regulated by NO, CO and H2S on top of the central role of O2 in regulating bioenergetics and metabolic pathways. Research by academic institutions as well as pharmaceutical companies are endeavoring to harness the beneficial effects of gas signals in order to treat a range of conditions including diabetes, transplant rejection, sepsis, atherosclerosis and cancer. Despite the scientific and clinical importance of dissolved gases, quantitative methods to measure real time effects of dissolved gases on tissue/cells are not available. Investigators who have studied the beneficial signaling initiated by trace gases and/or who are developing drugs to trigger the same benefits almost exclusively use water soluble surrogates/donors of each signaling gas. A critical point for this proposal is that such chemical donors of gases may not allow for accurate control of gas levels within tissue, and due to their low aqueous solubility, rapidly deplete from culture media. Preliminary data we have generated and published revealed opposite effects of dissolved H2S vs. that obtained with an aqueous provider of H2S, indicating a need to re- evaluation the effects of what is established regarding NO, H2S and CO from studies using donor molecules. As most life science researchers do not have the ability to study the direct effects of trace gases at physiologically relevant concentrations, we will develop a turnkey, automated instrumentation to control the concentration and exposure time of tissue to media containing user-specified levels of 6 gases including NO, CO, H2S, O2, CO2 and N2. This automated and user-friendly system will be constructed to supply gas mixtures to a variety of widely used tissue assessment modalities including fluidics systems, static culture in plates, and cuvette systems for studying gas binding interactions to purified proteins. We have assembled a team consisting of Bio- and Mechanical/Combustion Engineers with many years of experience designing and constructing gas fluidics system applied to biological analysis, as well as an Applied Mathematician, an Analytical Chemists and Cellular Physiologists who will be involved with the validation of the instrumentation. This technology will have broad impact on fundamental research by facilitating the study of kinetic and concentration-dependent effects of signaling gases on tissue function, signaling and bioenergetics, and evaluation of therapeutics being developed to mimic the cytoprotective properties of the gases.