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

• 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

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，活性氧已被牵连在各种疾病的发病机制。虽然活性氧中间体在药物代谢中的存在可以从终产物分析或从抗氧化剂或酶如超氧化物歧化酶的作用中推断，但只有电子自旋共振（ESR）技术允许直接检测这些高活性物质。然而，一些自由基物种由于其极短的半衰期而无法通过ESR检测到，这导致自由基的稳态浓度低或自由基弛豫时间短，这导致非常宽的线。这些事实使得近年来发展起来的自旋捕集和化学发光技术被广泛应用于自由基的检测。该项目的目标是开发各种可用于测量生物模型中活性氧的测定方法。在本研究计划中，我们希望建立（1）自旋捕获/ESR技术， ...更多信息 （2）该技术在荧光-光学生物传感器中的应用，该方法具有灵敏度高、可连续、在线、无损检测活性氧的优点。1.去铁胺（DFX）是一种过渡金属离子螯合剂，可用于H_2O_2和过氧化物酶的检测。我们证明DFX的单电子氧化产物DFX氮氧自由基是H_2O_2-过氧化物酶反应的中间体，因此，H_2O_2和过氧化物酶可被ESR波谱检测到（μ M）2.Fe（DTCS）_2配合物作为NO捕集剂的ESR波谱法可用于体外体系中NO的检测;然而，由于Fe（DTCS）_2的毒性或L-带ESR灵敏度低，用于检测活体NO的条件不是最佳的。3.利用自旋捕集剂2，2，6，6-四甲基哌啶（TEMP）对玫瑰红光活化产生单线态氧（^1O_2）的ESR谱进行了研究。ESR研究结果表明，虎红经光活化后产生^1O_2，并以^1O_2-TEMP产物（克里思; 2，2，6，6-四甲基哌啶-N-氧基）的形式被检测<-8>到<-4>。应该可以在多种条件下和在多种干预下测量氧衍生的自由基的产生。5.氧衍生的自由基，可能是羟基自由基，直接在犬牙齿的狗牙髓中产生血管收缩。这可以通过破坏基础释放的EDRF和/或内皮功能障碍来解释。少

项目成果

岡部 栄逸朗: "薬理学における生体フリーラジカル検出技術の最前線" 日本薬理学雑誌. 110・3. 132-135 (1997)

Eiichiro Okabe：“药理学中生物自由基检测技术的前沿”日本药理学杂志110・3（1997）。

Masahiro Norisue: "Inhibition by hydroxyl radicais of calcionin gene-related peptide-mediated neurogenic vasorelaxation in isolated canine lingual artery" Journal of Pharmacology and ExperimentalTherapeutics. 280・1. 492-500 (1997)

Masahiro Norisue：“羟基自由基对离体犬舌动脉中降钙素基因相关肽介导的神经源性血管舒张的抑制”《药理学和实验治疗学杂志》280・1（1997）。

Kawakami, M.et al.: "Superoxide anion radical-triggered Ca^<2+> release from cardiac sarcoplasmic reticulum though ryanodine receptor Ca^<2+> channel." Molecular Pharmacology. (in press). (1998)

Kawakami, M.et al.：“超氧阴离子自由基触发 Ca^<2> 通过兰尼碱受体 Ca^<2> 通道从心脏肌浆网释放。”

Okabe, E.et al.: "Myocardial ischemia-reperfusion injury : sarcoplasmic reticulum dysfunction depends on reactive oxygen species production." Magnetic Resonance in Medicine. 9. 48-51 (1998)

Okabe, E.等人：“心肌缺血再灌注损伤：肌浆网功能障碍取决于活性氧的产生。”

Lee, C.et al.: "A mechanism underlying free radical-mediated reduction of Ca, Mg-ATPase activity of skeletal muscle sarcoplasmic reticulum." Magnetic Resonance in Medicine. 6. 384-386 (1995)

Lee, C.等人：“自由基介导的骨骼肌肌浆网 Ca、Mg-ATP 酶活性降低的机制。”