隨著人口結構的老化以及肥胖人口的增加,心血管疾病對人類的健康傷害極大其致死率甚至超過癌症.其中,又以心肌梗塞占大多數,病患因冠狀動脈阻塞造成其下游的心肌細胞壞死,最終因心律不整而死亡.至目前為止,臨床上仍未能找出有效的方法來促進受損的心臟恢復.斑馬魚是近年來非常受歡迎的疾病模式生物,其優點包含胚胎上的透明性,能產生大的子代,已被解碼的基因遺傳訊息與人類有高度的相似性,並且有大量文獻使用斑馬魚來探討心血管相關疾病.在近期心臟再生的研究上,斑馬魚展現其獨特的修復能力,正因此項特性,我們希望利用斑馬魚來建立用於探討心肌梗塞與其治療發展的整合性平台.
在本篇研究,可分為四個部分.第一部份,我們建立一個心肌梗塞的斑馬魚模式用於探討心臟受損及修復的過程,並從細胞的層次進行觀測.第二部分,我們測試一套新穎的斑馬魚心電圖系統,用於量測斑馬魚受損之心臟電生理狀況.第三部分,我們結合血管造影技術及螢光轉殖基因魚,來探討斑馬魚受損之心血管循環恢復情形.第四部份,我們製備新的螢光轉殖基因魚,期望應用於藥物檢測.最終,我們希望整合這些平台並提供心肌梗塞上的研究與其治療方向.

Table of contents
中文摘要 I
Abstract II
致謝 IV
Abbreviations VII
1. Introduction 1
1.1 The cardiovascular system and its diseases 1
1.2 Zebrafish model for myocardial infarction 2
1.3 Cardiac regenerative capacity in zebrafish 3
1.4 Important events during cardiac repair in zebrafish 4
1.5 Advantages of zebrafish model for myocardial infarction research and therapeutics development 5
1.6 The objective of this study 6
2. Material and Methods 8
2.1 Zebrafish strains and transgenic fish lines stocks 8
2.2 Generation of new transgenic fish line 8
2.3 Cryoinjury 8
2.4 Histology and immunofluorescence staining 9
2.5 Angiography in adult zebrafish- retro orbital injection 9
2.6 Image capture and the use of microscope 10
3. Results 11
3.1 The adult zebrafish model of cryoinjury-induced myocardial infarction 11
3.1.1 Cryoinjury-induced myocardial infarction in zebrafish 11
3.1.2 Morphological change in the ventricle apex in response to cryoinjury 12
3.1.3 Extent of tissue damage following cryoinjury 12
3.1.4 Cryoinjury induces phagocytes recruitment in the lesion area 14
3.2 Assessing the extent of cardiac injury by electrocardiography 15
3.2.1 Functional analysis by a custom-made ECG kit and a digital camera 15
3.2.2 Prolonged ventricular re-polarization caused by infarction in zebrafish heart 16
3.3 Endothelial dynamics in response to myocardial infarction 17
3.3.1 Rapid recovery of the blood vessel endothelium in the infarcted ventricle 18
3.3.2 Blood reperfusion within the infarction site 19
3.4 Generation of transgenic zebrafish to report heart regeneration 20
3.4.1 Generation of Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) to report cardiomyocytes dedifferentiation in real time 21
3.4.2 F1 generation of Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) were identified by two-color fluorescent signals in the heart. 21
4. Discussion 23
4.1 Requirement of assay platform for the study of MI-like injury in zebrafish model 24
4.2 Endothelial dynamics – vessel re-growth and blood reperfusion 25
4.3 Application for drug screen 26
4.4 Future perspective 27
5. Reference 28

List of Figures 31
Fig1. Morphological change in the ventricle apex in response to cryoinjury 31
Fig2. Necrotic or apoptotic cells in the ventricle apex of cryoinjured heart 32
Fig3. Cryoinjury leads to loss of endothelial cells and cardiomyocytes. 33
Fig4. Phagocytes were recruited to the lesion site following cryoinjury 34
Fig5. Prolonged ventricular re-polarization caused by cryoinjury-induced infarction in zebrafish isolated heart. 35
Fig6. Rapid recovery of the cardiac endothelium in the infarction site upon cryoinjury 36
Fig7. Infarction region rapidly vascularized and re-perfused after cryoinjury 37
Fig8. Stable transgenic zebrafish line Tg(gata4:nls-mCherry/cmlc2:nls-EGFP) were identified by two-color fluorescent signals in the heart 38

List of supporting information 39
FigS1. Preparation of cryoprobe 39
FigS2. Phagocytes retained in the apex of ventricle at 7dpc following cryoinjury………..40
FigS3. Combination of a custom-made ECG kit and a digital camera for the assessment of heart function 41
FigS4. Tail vein vasculature and blood flow could be detected by angiography 42

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