在人體中，急性心臟損傷 - 例如心肌梗塞後的修復，涉及一系列複雜的組織重塑機制，使得纖維化疤痕會取代受損的心臟組織，進而影響心臟的收縮功能，並可能因此導致心臟衰竭而死亡。
相較於哺乳類動物，斑馬魚的心臟在受到嚴重損傷後可以進行完善的再生且不留疤痕。 這意味著我們能透過從斑馬魚心臟再生期間的調節機制中學習，並找出解決人類心臟纖維化的可能性。
先前的研究顯示在轉基因脊椎動物的心肌細胞，特異性過度表達transglutaminase-2時，可誘導其心室重構。因此，在本研究我們假設Tgm2可能是斑馬魚心臟再生過程中細胞外基質（ECM）重塑的關鍵調節因子。為了驗證這個假設，首先我們利用斑馬魚心臟創傷微陣列進行數據分析。我們發現tgm2b的基因表達，在成年斑馬魚心臟凍傷後顯著上調。我們接下來證實了冷凍損傷後，斑馬魚心肌Tgm2的酵素活性會相應的增加。為了探討Tgm2功能喪失的影響，我們使用TGM2抑制劑ERW1041E，來抑制心臟損傷後的斑馬魚Tgm2的酵素活性。AFOG染色顯示當Tgm2活性受到抑制時，傷口區域的膠原蛋白沉積減少。我們還發現col1a1a和tgfb1a的基因表達，會因為Tgm2活性受到抑制而共同降低。此外，Tgm2活性的喪失可能影響纖連蛋白的蛋白質表達。綜合實驗結果，Tgm2可能在斑馬魚心臟再生期間調節ECM蛋白的產生，因此Tgm2與其調控機制可能有潛力做為治療心臟纖維化的藥物標的。

In human, damage repair after acute cardiac injury involves a series of complex tissue remodeling events to replace damaged tissue with a fibrotic scar. In contrast, zebrafish heart can undertake complete regeneration after severe injury. This implies the possibility of resolving human cardiac fibrosis by learning from the regulatory mechanisms during zebrafish heart regeneration.
Previous studies have shown that cardiomyocyte-specific overexpression of transglutaminase-2 (TGM2) will induce ventricular remodeling in transgenic vertebrates. Therefore, this study hypothesized that Tgm2 may be a critical regulator of extracellular matrix (ECM) remodeling during zebrafish heart regeneration. To investigate this hypothesis, we performed data mining by microarray analysis. We found that tgm2b gene expression was significantly up-regulated after cardiac cryoinjury in adult zebrafish. We next verified that Tgm2’s enzymatic activity increased accordingly after cryoinjury. To study the loss-of-function effect, we used a potent TGM2 inhibitor ERW1041E to treat the zebrafish after cardiac injury. AFOG staining showed that the deposition of collagen in the wounded region was reduced when Tgm2 activity was inhibited. We also found that the expression levels of col1a1a and tgfb1a would decrease collectively by Tgm2 inhibition. Moreover, loss of Tgm2 activity might affect the protein expression of fibronectin. In conclusion, Tgm2 could regulate the production of ECM proteins during zebrafish heart regeneration, consequently, Tgm2 and its regulatory mechanism might be a therapeutic target for cardiac fibrosis.

中文摘要------------------------------------------------ i
Abstract----------------------------------------------- ii
致謝--------------------------------------------------- iii
Table of Contents-------------------------------------- iv
Abbreviations------------------------------------------ vi
1. Introduction------------------------------------------------ 1
1.1 Cardiovascular disease------------------------------------- 1
1.2 The advantages of zebrafish model for cardiac tissue repair research------------------------------------------------------- 1
1.2.1 Zebrafish as a model for cardiovascular diseases--------- 1
1.2.2 Heart regeneration in zebrafish-------------------------- 2
1.2.3 Cardiac injury model in zebrafish------------------------ 3
1.2.4 Staging of the zebrafish cardiac remodeling process------ 3
1.3 Extracellular matrix remodeling dynamics------------------- 4
1.4 Transglutaminase-2 and heart disease----------------------- 5
1.5 The specific aim of this study----------------------------- 6
2. Material and Methods---------------------------------------- 7
2.1 Zebrafish husbandry---------------------------------------- 7
2.2 Zebrafish anesthetization and ventricular cryoinjury------- 7
2.3 Transglutaminase activity assay---------------------------- 8
2.4 Transglutaminase 2 inhibitor------------------------------- 8
2.5 Rna isolation and real-time quantitative pcr analysis------ 9
2.6 Afog staining---------------------------------------------- 9
2.7 Immunofluorescence staining-------------------------------- 10
3. Results----------------------------------------------------- 11
3.1 The tgm2b gene expression and transglutaminase (tgm) activity profile after cryoinjury--------------------------------------- 11
3.1.1 The injury-induced tgm2b gene expression in zebrafish heart ----------------------------------------------------------------11
3.1.2 Upregulation of transglutaminase activity in zebrafish heart at 3dpci------------------------------------------------------- 12
3.1.3 tgm2 expression patterns after cryoinjury in the adult zebrafish heart------------------------------------------------ 13
3.2 ERW1041E is a potent inhibitor of transglutaminase-2 activity in zebrafish.-------------------------------------------------- 13
3.3 Transglutaminase-2 activity was required for collagen formation during zebrafish heart regeneration.----------------- 14
3.4 Transglutaminase 2 activity was required for the production of fibronectin during zebrafish heart regeneration---------------- 15
3.5 The correlation between tgm2 enzymatic activity and the known heart regeneration signaling pathways-------------------------- 16
4. Conclusion and Discussion----------------------------------- 18
4.1 Proposal model for the role of tgm2 in zebrafish heart repair after cryoinjury----------------------------------------------- 18
4.2 Lessons from zebrafish heart generation may help to accelerate tissue remodeling in human heart disease patients-------------- 19
4.3 Characterization of syndecan-4 and transglutaminase-2 in adult zebrafish heart regeneration.---------------------------------- 20
5. Reference--------------------------------------------------- 22

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