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，使溶解气体的研究能够 生物样本。血气(O2和CO2)以及微量信号气体(NO、CO和H2S)都起作用 在调节多种组织功能方面起着关键作用，如心率、血流量、 免疫反应、激素和神经递质分泌，以及细胞保护和抗炎 属性。事实上，大多数组织(如果不是所有的话)都会产生并受到中央顶端的NO、CO和H2S的调节 氧在调节生物能量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影响，以治疗 一系列疾病，包括糖尿病、移植排斥反应、脓毒症、动脉粥样硬化和癌症。尽管 溶解气体的科学和临床重要性，测量实时影响的定量方法 组织/细胞上的溶解气体不可用。研究了有益信号的研究人员 由痕量气体发起和/或正在开发药物以触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。这项提议的一个关键点是，这种化学物质 气体供体可能不允许精确控制组织内的气体水平，并且由于其低含水 溶解度，从培养基中迅速耗尽。我们生成并发布的初步数据显示 溶解的硫化氢与使用含水的硫化氢供应商获得的相反的影响，表明需要重新 评估从使用供体分子的研究中确定的关于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在 生理上相关的浓度，我们将开发一种交钥匙的自动化仪器来控制 组织在含有用户指定水平的6种气体的介质中的浓度和暴露时间 包括NO、CO、H_2S、O_2、CO和N_2。这个自动化和用户友好的系统将被构建为 向各种广泛使用的组织评估方式提供气体混合物，包括流体系统、静态 平板培养和试管系统，用于研究气体与纯化蛋白质的相互作用。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 设计和构建用于生物分析的气体射流系统，以及一个实用的 数学家、分析化学家和细胞生理学家将参与验证 仪器仪表。这项技术将对基础研究产生广泛的影响，促进 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效应， 以及正在开发的治疗方法的评估，以模拟气体的细胞保护特性。

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.