人類心肌梗塞後的損傷修復牽涉到複雜的機制，在修復的過程中，受傷的心臟組織會由膠原蛋白等成份組成的纖維化傷疤所取代。這些不具收縮能力的纖維化組織會對心臟的肌肉收縮造成長久的影響，嚴重時更可能會造成心肌衰竭而死亡。相較於哺乳類動物，斑馬魚擁有優秀的修復能力且能夠在心肌受損後進行無疤再生。這意味著在斑馬魚心臟再生過程中可能具有獨特的傷疤調控機制。為此我們利用斑馬魚心室冷凍創傷模型來研究是什麼因子可參與調控受損心臟傷疤的生成與退減。
先前的研究顯示，在高等脊椎動物中，syndecan-4在調節發炎反應和心肌成纖維細胞 (fibroblasts) 的功能中扮演著重要的角色。有趣的是，我們實驗室先前針對斑馬魚心臟創傷後再生過程的微陣列分析也指出Sdc4可能參與了心臟修復的過程。因此，我們假設Sdc4可能在斑馬魚心臟創傷後心臟修復與傷疤形成中扮演關鍵的作用。為了驗證這一個假設，我們首先進行斑馬魚心臟創傷的基因分析，結果顯示在成年斑馬魚心臟創傷後，sdc4 的基因表現如預期快速上升。接下來，我們透過注射預混合的siRNA和SilenceMags奈米磁珠到血管中，再以心臟上方外加磁場的方式，建立在斑馬魚心臟抑制sdc4基因表現的動物模型。AFOG組織染色檢驗顯示當sdc4基因被抑制時，斑馬魚心臟傷疤面積會減少，代表Sdc4參與膠原蛋白的沈積。除此之外，我們發現當sdc4基因被抑制時，mmp9 和mmp2的基因表現上升，這代表Mmp9 和 Mmp2可能參與在Sdc4調控的細胞外間質重塑。最後，我們發現sdc4會影響tgfb1a訊息傳遞路徑，這可能說明特殊的sdc4- tgf-β訊息傳遞路徑與斑馬魚的心臟無疤再生有關。綜合上述結果，Sdc4可能是斑馬魚心臟再生過程中調控傷疤生成與減少的關鍵角色。

The wound healing process after human myocardial infarction (MI) involves complex events to replace damaged tissue with a collagen-rich fibrotic scar, in which the muscular contraction of the affected cardiac tissue may loss its function permanently. In contrast, zebrafish displays a powerful capacity of scar-free regeneration post-cardiac injury. This implies that heart regeneration of zebrafish must have a unique mechanism to resolve the scarring retention issue. We thus adapted the ventricular cryoinjury model in this study to investigate what factors regulate scar formation and reduction during zebrafish heart regeneration. Previous studies have revealed that syndecan-4 (SDC4) plays a crucial role in regulating cardiac fibroblasts and inflammation following injury in higher vertebrates. Interestingly, our zebrafish heart regeneration microarray data indicated Sdc4 may associate with these processes. Therefore, we hypothesized that Sdc4 may be differentially regulated in scar-free repair during zebrafish heart regeneration. To investigate this hypothesis, we first conduced an expression analysis to check whether sdc4 and scar associated genes were up-regulated after heart injury in adult zebrafish. Next, we induced heart specific sdc4-knockdown via siRNA and SilenceMag magnetofection technology which enclaved the zebrafish heart with a forced magnetic field. As expected, we found decreased sdc4 expression corresponded well to zebrafish heart scar retention by AFOG staining, in which collagen deposition was inhibited. Moreover, we found mmp9 and mmp2 were also up-regulated, which supports Sdc4’s role in ECM remodeling and regression. Finally, we found tgfb1a expression was linked with sdc4 regulation, which supports the involvement of a unique Sdc4-Tgf-β signaling axis in the scar-free healing of zebrafish heart. In conclusion, Sdc4 could be a critical regulator for scar formation and retention during zebrafish heart regeneration.

中文摘要 I
Abstract II
致謝 III
Table of contents IV
Abbreviations VIII
1. Introduction 1
1.1 Cardiovascular disease 1
1.2 Zebrafish as a model organism for human diseases 1
1.3 Heart regeneration in zebrafish 2
1.3.1 High regeneration capacity 2
1.3.2 Cardiac injury model in zebrafish 3
1.3.3 Advantages of conducting heart research in zebrafish 3
1.3.4 Zebrafish heart remodeling process 4
1.4 ECM protein remodeling dynamics 5
1.5 Syndecan-4 and heart disease 6
1.6 The specific aim of this study 6
2. Material and methods 8
2.1 Zebrafish husbandry 8
2.2 Zebrafish mating and microinjection 8
2.3 Zebrafish anesthetization and ventricular cryoinjury 8
2.4 Adult zebrafish retro-orbital injection 9
2.5 Inducing sdc4 knockdown of the heart in adult zebrafish 9
2.6 RNA isolation and Real-time quantitative PCR analysis 10
2.7 AFOG staining 10
2.8 Immunofluorescence staining 11
3. Results 12
3.1 The zebrafish model of cryoinjury-induced myocardial infarction 12
3.1.1 Cryoinjury to the ventricle of adult zebrafish heart 12
3.1.2 Scar formation during zebrafish heart regeneration 13
3.2 Heart-specific gene silencing in vivo - Establishment of SilenceMag mediated sdc4 knockdown in the adult zebrafish heart 14
3.2.1 Dynamic regulation of sdc4 during adult zebrafish heart repair 14
3.2.2 Validation of sdc4 siRNA efficiency in zebrafish embryos 15
3.2.3 In vivo SilenceMag induced sdc4 knockdown in adult zebrafish heart 16
3.3 The role of sdc4 in collagen scar formation during heart regeneration in zebrafish 16
3.3.1 Inhibition of sdc4 upregulation reduced collagen deposition 17
3.3.2 Inhibition of sdc4 upregulation impaired the production of ECM proteins 18
3.4 MMPs expression analysis after cryoinjury 19
4. Conclusion and Discussion 21
4.1 Establishment of novel Sdc4 deficiency adult zebrafish heart injury model 21
4.2 Proposal model for the role of Sdc4 in zebrafish heart repair after cryoinjury 22
4.3 Lessons from zebrafish heart generation may help to reduce scarring in human heart disease patients 23
5. Other achievement 25
5.1 Translational application - IRB application outline 25
SDC4 as a novel marker to predict severity of AMI and left ventricular functions 25
6. Reference 27

List of Tables
Table. 1 The zebrafish siRNA sequences list 31
Table. 2 Primer list of Real Time-quantitative PCR analysis 32

List of Figures
Fig. 1 Cryoinjury tool and cryoinjury procedure 33
Fig. 2 Scar formation and regression during healing after ventricular cryoinjury. 34
Fig. 3 Verification of sdc4 siRNA efficiency by real-time PCR. 35
Fig. 4 Inducing sdc4 knockdown of the heart in adult zebrafish. 36
Fig. 5 Inhibition of sdc4 preserves zebrafish heart regeneration. 37
Fig. 6 Inhibition of sdc4 increase the degradation of blood clot in the post-infarct. 38
Fig. 7 Inhibition of sdc4 increase the degradation of ECM proteins in the post-infarct. 39
Fig. 8 Target gene expression analysis of the 3 dpci infarcted heart. 40
Fig. 9 Altered mmp9 and mmp2 gene expression on the 7 dpci infarcted heart. 41
Fig. 10 Proposal model for zebrafish heart repair after cryoinjury. 42

Supporting Information
Fig. S1 Target gene expression profiling during zebrafish heart repair. 43
Fig. S2 The sdc4 gene expression profiling during heart repair after cryoinjury 44

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