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研究生： 林旻萱
Lin, Min-Hsuan
論文名稱： 模式生物斑馬魚的心電圖檢測平台應用於藥物篩檢及電生理研究
Zebrafish in vivo electrocardiogram platform for drug assay and electrophysiology research
指導教授： 莊永仁
Chuang, Yung-Jen
口試委員: 吳金烈
Wu, Jen-Leih
張壯榮
CHANG, CHUANG-RUNG
劉旺達
Liu, Wangta
魯才德
Lu, Tsai-Te
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2019
畢業學年度： 107
語文別： 英文
論文頁數： 53
中文關鍵詞： 斑馬魚 、心電圖 、心律不整 、藥物篩檢
外文關鍵詞： zebrafish, electrocardiography, arrhythmia, drug assay
相關次數： 點閱：2  下載：0 
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    • 斑馬魚是一個生醫研究的重要模式生物。近年來越來越多與心血管疾病相關的研究，使用成年斑馬魚來模擬疾病生理，與藥物機制探討。其中，心電圖分析在斑馬魚中的應用，已成為心臟生理學、毒物學及藥物篩選研究的重要工具。由於現今市面上，可偵測記錄活體成年斑馬魚心電圖的系統，成本偏高且操作限制多，僅少數實驗室才有能力執行相關的研究。在本研究中，我們整合開發一個經濟的活體斑馬魚心電圖偵測平台，並針對平台操作進行了優化。此平台包括攜帶型心電圖套件與一組客製的三相針式電極，接著以此平台進行給予斑馬魚藥物，並測量藥物對其心電圖的影響。我們首先測試了複合型麻醉劑 MS-222 及 Isoflurane 對斑馬魚心律的作用，確定最佳測試實驗條件，以降低麻醉對斑馬魚心臟生理訊號的影響。我們接著選擇數種已知會影響人類心律的藥物進行測試，包括使加速心跳的 isoproterenol，和降低心律或影響 QT interval 的抗心律不整藥物： verapamil、quinidine 及 amiodarone，以及具神經毒性的 veratridine。實驗結果發現，這些心臟藥物在成年斑馬魚中誘導出的電生理反應，與人類的反應具有高度的相似性，也驗證此平台的實用性。我們認為這套活體成年斑馬魚心電圖平台，因為具有操做的便利性，所以不僅能進行斑馬魚的心臟生理與相關藥物的研究，也能用以推動電生理學的相關教學，可望促進未來心血管基礎研究的轉化應用。

    Zebrafish is a popular and favorable model organism for cardiovascular research, with an increasing number of studies implementing functional assays in the adult stage. For example, the application of electrocardiography (ECG) in adult zebrafish has emerged as an important tool for cardiac pathophysiology, toxicity, and chemical screen studies. However, few laboratories are able to perform such functional assay due to the high cost and limited availability of a convenient in vivo ECG recording system. In this study, an inexpensive ECG recording platform and operation protocol that has been optimized for adult zebrafish ECG research was developed. The core hardware includes integration of a ready-to-use portable ECG kit with a set of custom-made needle electrode probes. A combined anesthetic formula of MS-222 and isoflurane was first tested to determine the optimal assay conditions to minimize the interference to zebrafish cardiac physiology under sedation. For demonstration, we treated wild-type zebrafish with several pharmacological agents known to affect cardiac rhythms in humans, including isoproterenol, verapamil, quinidine, amiodarone and veratridine. Conserved electrophysiological responses to these drugs were induced in adult zebrafish and recorded in real time. The results show that zebrafish have highly similar physiological responses with human after most drug treatments. This economic ECG platform has the potential to facilitate teaching and training in cardiac electrophysiology with adult zebrafish and to promote future translational applications in cardiovascular medicine.

    中文摘要 I Abstract II Abbreviations III Cardiac related abbreviation and translation IV Table of contents V Preface VII 1. Introduction 1 1.1 Analysis of Electrocardiography (ECG) 2 1.2 Animal models for research 3 1.3 Zebrafish ECG recording 4 1.4 Zebrafish in ECG studies 4 1.5 Drugs induced cardiac arrhythmias 6 1.6 Specific aim of this study 7 2. Materials and methods 8 2.1 Zebrafish 9 2.2 Anesthetics preparation 9 2.3 Electrode probes 9 2.4 ECG kit 10 2.5 ECG signal processing 10 2.6 Drug treatment 11 2.7 Statistical analysis 12 3. Results 13 3.1 Constructing the adult zebrafish ECG system 14 3.2 ECG recording of adult zebrafish 15 3.3 Electrocardiography of adult zebrafish 16 3.4 Electrocardiography of zebrafish under prolonged sedation 16 3.5 Effect of isoproterenol treatment on drug-induced bradycardia 18 3.6 Induction of bradycardia under verapamil treatment 19 3.7 Effects of amiodarone on heart rate, QRS interval, QT interval, PR interval and QTc interval 19 3.8 Prolongation of QTc after quinidine treatment 20 3.9 Veratridine induce AV-block in adult zebrafish 21 4. Discussion 22 4.1 The detail of the ECG platform 23 4.2 The anesthetic formula we used in this study 24 4.3 This platform could detect the ECG of mouse and various type of small fish 25 4.4 Drugs that cause QT prolongation and/or TdP 25 4.5 QT interval measurement: Fridericia formula was used in this study 26 4.6 The limitation and improvement of this system 27 4.7 Perspective of this study 28 5. References 29 6. Figures 37 Figure 2. The results of Signal-to-noise ratio evaluation for EzInstrument’s ECG Kit. 39 Figure 3. Illustration showing the system setup and the three-needle electrode placement during real-time adult zebrafish ECG recording 40 Figure 4. An ECG recording of adult zebrafish with the three-needle electrodes 41 Figure 5. Effects of anesthetics: MS-222 alone and MS-222/isoflurane combination and prolonged sedation with combined formula on heart rate 42 Figure 6. Variation of heart rate during real-time recording of adult zebrafish and the response to isoproterenol 43 Figure 7. Real-time ECG recording of adult zebrafish and the response to verapamil treatment 45 Figure 8. Adult zebrafish ECG features and drug response to amiodarone 46 Figure 9. Quinidine reduced heart rate and prolonged QTc interval in adult zebrafish heart 47 Figure 10. Arrhythmia was induced after veratridine treatment in adult zebrafish 48 Figure 11. Mouse ECG recording was performed 49 Figure 12. Various type of small fish ECG recording was performed 50 Figure 13. Quinidine induced high-degree AV blocks 51 7. Appendix table 52 Figure A1. Different zebrafish physiological detection devices and their ECG results by ECG kit 53

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