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研究生:簡亦隆
研究生(外文):Chien, Yi-Lung
論文名稱:可同步量測斑馬魚於缺氧再灌流模式下腦部組織氧濃度與NADH變化之光學感測系統開發
論文名稱(外文):Development of an optical detection system for simultaneously measuring tissue oxygenation and NADH variation in zebrafish brain during Hypoxia / reperfusion
指導教授:黃士豪黃士豪引用關係
指導教授(外文):Huang, Shih-Hao
口試委員:莊昀儒吳志偉
口試委員(外文):Zhuang, Yun-RuWu, Chih-We
口試日期:2016-01-27
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:機械與機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:54
中文關鍵詞:磷光相位系統斑馬魚幼魚組織氧濃度NADH自體螢光DMD系統
外文關鍵詞:phase-based phosphorescence lifetime detection systemjuvenile fish of zebrafishtissue oxygenationNADH fluorescent intensityDMD sensing system
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於先前的研究,我們已開發出可量測單一斑馬幼魚體內組織血氧分佈之光學裝置,成功的量測在環境氧分壓極端變化下,魚體內循環谷與尾靜脈等組織的血氧濃度。而本研究將進一步討論在急性缺氧/再灌流對於斑馬幼魚腦部的影響,藉由量測腦部組織的氧濃度以及NADH的變化來評估缺氧/再灌流對魚體所造成的損傷。本研究利用磷光相位差光學檢測裝置整合微流體晶片,開發出可檢測單一斑馬幼魚於缺氧/再灌流模式下腦部血氧與NADH變化之光學感測系統。
此光學感測系統結合了兩個部分組件:斑馬魚定位微流體晶片與磷光相位差光學檢測系統。斑馬魚微流體晶片:設計定位微流體晶片可讓斑馬魚定位於微流道中並可長時間培養,且可藉由注入的不同氧氣濃度的培養液來改變流道內環境的氧分壓,藉由蠕動幫浦帶動流體持續注入培養液並帶走廢液以保持環境對魚體穩定的刺激。磷光相位差光學檢測系統:透過投影機內的Digital Micromirror Device (DMD),來控制檢測光源的光源投影圖形,激發欲檢測之區域組織內的磷光染劑與NADH自體螢光,並經由電腦控制,達到自動化的即時激發量測效果。
本研究使用(Dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate)以下簡稱為Ru(Phen)做為斑馬魚幼魚體內的磷光感測物質,並利用顯微注射技術注入到受精後48小時的魚體,以常見的緩衝溶液Phosphate-buffered saline(PBS)來稀釋濃度,並針對不同濃度與注射量做存活測試,最後採用Ru(Phen)注射液的濃度為4mM、體積為9.2nl,來建立腦部的感測系統。
本研究使用利用DMD光學架設投影不同圖形的激發光於斑馬魚的腦部,成功量測到前腦(Forebrain)與中腦(Midbrain)的組織氧含量與NADH螢光。這項實驗我們討論了兩個實驗參數;缺氧時間與缺氧濃度,缺氧濃度定為0%與3.8%,我們觀察到缺氧10min後斑馬魚腦部的氧濃度下降到與外在缺氧環境相同,所以我們定缺氧時間為15min與30min,回復時間15min,持續缺氧後15min後再灌入16%的常氧水回復15min,組織氧濃度並未回升至基準值,且隨著缺氧時間的拉長(30min)而有所降低,而在不同的缺氧濃度(0%、3.8%)同樣缺氧時間,缺氧濃度0%的組織氧濃度回復值比3.8%的更低,組織氧濃度的回復情形隨著缺氧時間(15min、30min)的提升與缺氧濃度(0%、3.8%)的降低而有所降低。NADH螢光強度在缺氧過程中持續增加,在相同缺氧時間條件下,缺氧濃度越高NADH增加的越多。缺氧15min後 NADH螢光強度仍持續增加並未達到峰值,再灌流回復常氧15min,螢光強度快速下降,約3min螢光強度下降到穩定值,但比初始值來的高,且隨著缺氧時間的拉長(30min)而提升,而缺氧濃度越低(0%、3.8%)NADH的螢光強度增加的越多,其回復情形隨著缺氧時間的提升與缺氧濃度的降低而有所提升。這與組織氧濃度趨勢相反,為在缺氧過程中,斑馬魚體內粒線體因為組織氧濃度降低而導致電子傳遞鏈功能下降,當近乎無氧狀態時(組織氧含量濃度),電子傳遞鏈喪失功能,無法氧化NADH而產生ATP,導致NADH在細胞內濃度提升(螢光強度增強),而再灌流復氧階段,組織氧濃度的回升使電子傳遞鏈回復功能,快速消耗NADH產生APT以維持細胞運作,這時螢光因為NADH被消耗而降低,但無法回復水平,推測為缺氧時導致粒線體功能受損,電子傳遞鏈活性降低,NADH無法被消耗至正常水平。

In previous study, we have developed an optical device that can measure in vivo tissue oxygenation of cardiac region and cardinal vein of zebrafish embryo. In this study, we will further discuss the variation in zebrafish brain during acute hypoxia / reperfusion by measuring the change of oxygenation and NADH in brain tissue and to assess the hypoxia / reperfusion injury
The optical sensing system combines two part components: microfluidic chip and phase-based phosphorescence lifetime detection system. Microfluidic chip: design microfluidic chips allow zebrafish positioned and culture in tapered flow path and culture medium may be injected different dissolved oxygen in the flow path to change the environment. Phase-based phosphorescence lifetime detection system: a digital light modulation system that utilizes a modified commercial projector equipped with a light-emitting diode (LED) as a light source for quantitative measurements of in vivo tissue oxygenation in a zebrafish via phase-based phosphorescence lifetime detection.
The oxygen-sensitive phosphorescent probe (Ru(phen)) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish via the circulation valley to rapidly disperse probes throughout the embryo.
In this study, we developed a digital light modulation system that utilizes a modified commercial DMD projector equipped with LED as a light source to modulate the excitation light in the spatial and temporal domains for quantitative measurements of brain tissue oxygenation in a zebrafish in real time via phase-based phosphorescence lifetime detection. We have successfully measured in tissue oxygenation changes and NADH fluorescent intensity in the forebrain and midbrain of a 90 hpf zebrafish during hypoxia / reperfusion.

摘要 i
Abstract iii
目錄 iv
圖目錄 vi
表目錄 viii
第一章緒論 1
1.1前言 1
1.2研究背景 2
1.2.1缺血(氧)再灌流傷害 2
1.2.2細胞呼吸作用 3
1.2.3氧氣測量 4
1.2.4 NADH 4
1.2.5模式動物定位 5
第二章 文獻回顧 7
2.1體內氧濃度量測 7
2.2環境氧氣對斑馬魚的影響 12
2.3缺氧/再灌流 13
2.4顯微定位技術 14
2.5實驗動機與目的 15
第三章 實驗原理 17
3.1磷光相位差檢測原理 17
3.2磷光壽命檢測原理 19
第四章 實驗設計與架設 21
4.1實驗設計與機制 21
4.2磷光材料 22
4.3微流體晶片製作與設計 22
4.4磷光相位差光學檢測系統 23
4.5斑馬魚胚胎飼養裝置 25
4.6斑馬魚與胚胎 26
4.7 DMD系統 27
4.8斑馬魚幼魚與顯微注射 28
第五章 實驗結果與討論 32
5.1斑馬魚幼魚磷光感測物質注入存活測試 32
5.2 Ru(Phen)磷光粒子 34
5.3斑馬魚幼魚腦部光學檢測 35
第六章 結論於未來展望 42
6.1結論 43
6.2未來展望 44
參考文獻 45
附件 48

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[23] Kopp R, Bauer I, Ramalingam A, Egg M, Schwerte T (2014) Prolonged Hypoxia Increases Survival Even in Zebrafish (Danio rerio) Showing Cardiac Arrhythmia. PLoS ONE 9(2): e89099. doi: 10.1371/journal.pone.0089099

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