Development and Application of Bioradical Detecting System for Oral Tissues : A Basic Research

口腔组织生物自由基检测系统的开发与应用：基础研究

基本信息

批准号：
07557119
负责人：
OKABE Eiichiro
金额：
$ 1.15万
依托单位：
Kanagawa Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557119/
关键词：
Free radicals Electron spin resonance Microcirculation Nitric oxide Oral tissues Spin-trap エンドトキシン

项目摘要

In recent years, reactive oxygen species have been implicated in the pathogenesis of a wide variety of disorders. Although the existence of reactive oxygen intermediates in drug metabolism can be inferred from end product analysis or from the effects of antioxidants or enzymes such as superoxide dismutase, only the technique of electron spin resonance (ESR) allows the direct detection of these highly reactive species. However, some free radical species cannot be detected by ESR due to their extremely short half-lives, which result in low steady-state concentrations of the radicals or to short radical relaxation times, which lead to a very broad line. These facts made recent development of spin-trapping and chemiluminescence techniques are widely used to detect free radicals. The goal of this project is to develop the various assays available for measurement of reactive oxygen species in biological models. In this research project, we wish to establish (1) the spin-trapping/ESR techniqu … More e in vitro and vivo and (2) the chemiluminescence-optical biosensor application of this technique, a very sensitive method that has the advantage of being able to provide continuous, online, nondestructive monitoring of reactive oxygen species.1.Desferioxamine (DFX), a transition metal ion chelator, has been used to detect H_2O_2 and peroxidase enzyme. We demonstrated that DFX's one-electron oxidation product, DFX nitroxide free radical, is an intermediate of H_2O_2-peroxidase reaction ; thus, H_2O_2 and peroxidase enzyme are detected (-muM) with ESR spectroscopy (by detecting DFX radical).2.ESR spectrometry method using NO-trapping reagent, Fe (DTCS) _2 complex, may be useful for detecting NO in vitro system ; however, the conditions used for NO detection in vevo are not optimal due to Fe (DTCS) _2 toxicity or to low L-band ESR sensitivity.3.Generation of singlet oxygen (^1O_2) from photoactivation of rose bengal was studied by ESR spectroscopy by use of the spin-trap 2,2,6,6-tetramethylpiperidine (TEMP). The result of ESR study showed that ^1O_2 was produced by photoactivation of rose bengal and was detected as ^1O_2-TEMP product (TEMPO ; 2,2,6,6-tetramethylpiperidine-N-oxyl).4.Optical biosensor was developed for the batch analysis system of hypoxanthine in the concentration range between 10^<-8> and 10^<-4> M.With this optical biosensor, it should be possible to measure oxygen-derived free radical generation in a variety of conditions and with multiple interventions.5.Oxygen-derived free radicals, possibly hydroxyl radical, directly produces vasoconstriction in the dog dental pulp of canine teeth. This could be explained by the destruction of basally released EDRF and/or endothelial dysfunction. Less

近年来，活性氧与多种疾病的发病机制有关，尽管可以从最终产物分析或抗氧化剂或酶（例如超氧化物歧化酶）的作用推断出药物代谢中活性氧中间体的存在。电子自旋共振 (ESR) 技术可以直接检测这些高反应性物质，但是，某些自由基物质由于半衰期极短，导致自由基的稳态浓度较低，因此无法通过 ESR 检测。或者自由基弛豫时间短，导致谱线非常宽，这些事实使得自旋捕获和化学发光技术的最新发展被广泛用于检测自由基。该项目的目标是开发可用于测量反应性的各种测定方法。在这个研究项目中，我们希望建立（1）体外和体内自旋捕获/ESR技术以及（2）该技术的化学发光-光学生物传感器应用，这是一种非常灵敏的技术。该方法的优点是能够提供连续、在线、无损的活性氧监测。1.去铁胺(DFX)是一种过渡金属离子螯合剂,已被用来检测H_2O_2和过氧化物酶。我们证明了DFX的单-检测。电子氧化产物DFX硝基氧自由基是H_2O_2-过氧化物酶反应的中间体，因此，H_2O_2和用ESR光谱法检测过氧化物酶(-μM)(通过检测DFX自由基)。2.使用NO捕获剂Fe(DTCS)_2复合物的ESR光谱法可用于体外系统中检测NO。由于 Fe (DTCS) _2 毒性或 L 波段 ESR 灵敏度低，用于 vevo 中 NO 检测的方法并不是最佳选择。3. 产生单线态氧 (^1O_2)利用自旋阱2,2,6,6-四甲基哌啶(TEMP)通过ESR光谱研究了玫瑰红的光活化作用。ESR研究结果表明^1O_2是由玫瑰红的光活化产生的，并被检测为^。 1O_2-TEMP产物(TEMPO;2,2,6,6-四甲基哌啶-N-氧基)。4.开发光学生物传感器用于浓度范围在10^<-8>和10^<-4>M之间的次黄嘌呤的批量分析系统。利用这种光学生物传感器，应该可以测量各种条件下氧自由基的产生，并5.氧自由基（可能是羟基自由基）直接在犬齿牙髓中产生血管收缩，这可以通过破坏基础释放的 EDRF 和/或 EDRF 来解释。内皮功能障碍较少。