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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Ching-Min Chang | en |
| dc.contributor.author | 張靖敏 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:21:55Z | - |
| dc.date.copyright | 2010-07-12 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-07 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22599 | - |
| dc.description.abstract | 活性氧化物(reactive oxygen species, ROS)為具有高度氧化力的分子。ROS之生成可由細胞內或細胞外物質所誘發。當細胞內的ROS過量時會攻擊DNA、蛋白質和細胞膜脂質而造成無法修復的傷害，此與癌症、老化及疾病生成有關。在所有的活性氧化物中，過氧化氫 (H2O2) 被認為是最主要的分子，因為過氧化氫除了調節致病的機轉之外，目前已經有研究指出，過氧化氫也是一些訊號傳導路徑中的二級傳訊者。然而在多樣性的生物環境中，研究有關活性氧化物的過程一直遭遇一些無法克服的挑戰；主要的原因是因為在活體系統中，缺少對於活性過氧化物的專一性探針及研究方法。
在本篇研究中，我們首要目的是設計兩種化學螢光物質，我們稱之為探針一與探針二。這兩個探針都可以對過氧化氫有專一性的反應，並釋放出藍色的螢光物。因此我們可以利用此探針，在活體細胞中偵測出細胞面對壓力時所釋放出的過氧化氫。我們希望利用此篇研究的方法，建立一個簡單、快速、直接的平台，來偵測活化的巨噬細胞內之過氧化氫濃度;並藉由螢光儀偵測巨噬細胞內的相對螢光強度來定量。此外在未來的應用上，也可以利用此平台來尋找具有抗氧化劑活性的潛在化合物。 | zh_TW |
| dc.description.abstract | One major contributor to oxidative damage is hydrogen peroxide (H2O2), which has been considered mostly as reactive oxidative species (ROS), and mediate pathogenic processes. In addition, H2O2 is emerging as a newly recognized as a secondary messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems specifically. Here, we reported the synthesis and application of Probe 1 (1a and 1b), two new fluorescent probes that showed high selectivity for H2O2 and were capable of visualizing the addition of exogenous H2O2 to living cells. These probes were able to serve as a tool for evaluating the H2O2 production in complex physiological and pathological processes. The main purpose of this study was to create an easy, time-saving, and a straightforward platform for measurement the exact H2O2 concentration in living RAW 264.7 cell. Through quantitating the relative fluorescence intensity in vivo by spectrofluoromerty, we might discover new small molecules for potential antioxidants in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:21:55Z (GMT). No. of bitstreams: 1 ntu-99-R97b46032-1.pdf: 3377328 bytes, checksum: b0710b802ba2f7f139b458e022be816a (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | Table of Contents
中文摘要………………………………………………………………………………Ι Abstract………………………………………………………………………………..Ⅱ List of Figures…………………………………………………………………………Ⅲ List of Abbreviations………………………………………………………………….Ⅳ 1. Introduction 1 1.1 ROS (reactive oxygen species) 1 1.2 Hydrogen peroxide (H2O2) 2 1.2.1 Biochemical pathways of hydrogen peroxide generation and metabolism 2 1.2.2 The physiologic intracellular concentrations of hydrogen peroxide 6 1.2.3 Functions of hydrogen peroxide in biology 8 1.2.3.1 Stress or signaling 8 1.2.3.2 H2O2-dependent oxidative biosynthesis 10 1.3 Probes for H2O2 12 1.4 The criteria of designing effective chemical imaging probes for cellular hydrogen peroxide 14 1.5 Aim of this research 15 1.5.1 To develop and synthesize specific hydrogen peroxide probes 16 1.5.2 Measurement of H2O2 inside cells 19 2. Material and methods 21 2.1 Instrument and general information 21 2.1.1 Instrument 21 2.1.2. Reagents 22 2.2 General methods of organic synthesis 23 2.2.1 Synthetic route 23 2.2.2 Synthetic procedure 24 2.3 Bioassay 30 2.3.1 Materials 30 2.3.2 Analysis the spectrum property of probe 1 (1a and 1b) 31 2.3.3 Fluorescence responses of Probes to various reactive oxygen species (ROS) 31 2.3.4 Cell culture 32 2.3.5 Determination of small molecules on H2O2 production in living RAW 264.7 by fluorescence multiwall plate reader 32 2.3.6 Measure antioxidant effect of N-Acetylcysteine in LPS-stimulated RAW 264.7 cells by fluorescence multiwall plate reader 33 2.3.7 Preparation and Staining of Cell 34 2.3.8 Fluorescence Imaging Experiments 34 3. Results 35 3.1 Spectroscopic Properties and Optical Responses to H2O2. 35 3.2 Time-course of the fluorescence response of probes to H2O2 38 3.3 Fluorescence responses of probes to different reactive oxygen species 40 3.4 Fluorometric determination of intracellular H2O2 production induced by LPS in RAW264.7 cell 42 3.5 Antioxidant N-acetylcysteine (NAC) effect in living RAW264.7 cells stimulated by LPS 46 3.6 Fluorescence Image capturing of H2O2 in Living Cells using Confocal Microscopy 48 4. Discussion 53 4.1 The limitation of Probes’ Spectroscopic Properties 53 4.2 The specificity and senstivity of the probes 54 4.3 The fluorescense response of LPS-induced RAW 264.7 cells 57 4.4 The antioxidant effect of N-acetylcystenine (NAC) 57 4.5 Confocal fluorescence images of H2O2 in RAW 264.7 cells 58 4.6 Recommendation for future research 59 4.6.1 Improve the limitation of the research 59 4.6.2 Application 59 | |
| dc.language.iso | en | |
| dc.subject | 過氧化氫 | zh_TW |
| dc.subject | cellular signal transduction | en |
| dc.subject | second messenger | en |
| dc.subject | H2O2 scavenging molecule | en |
| dc.subject | hydrogen peroxide | en |
| dc.subject | antioxidants | en |
| dc.subject | spectrofluoromerty | en |
| dc.subject | reactive oxidative stress | en |
| dc.title | 以標的螢光探針研究巨噬細胞內之過氧化氫 | zh_TW |
| dc.title | Target Fluorescent Probes for Detecting Hydrogen Peroxide in Macrophage Cell | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 花國鋒(Kuo-Feng Hua),羅禮強(Lee-Chiang Lo) | |
| dc.subject.keyword | 過氧化氫, | zh_TW |
| dc.subject.keyword | hydrogen peroxide,reactive oxidative stress,second messenger,cellular signal transduction,H2O2 scavenging molecule,spectrofluoromerty,antioxidants, | en |
| dc.relation.page | 85 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-07-07 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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