Epithelial-to-mesenchymal transition in zebrafish epicardium is associated with inflammatory response during heart regeneration

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研究生：	余柏廷 Yu, Bo Ting
論文名稱：	Epithelial-to-mesenchymal transition in zebrafish epicardium is associated with inflammatory response during heart regeneration 發炎反應對於斑馬魚心外膜細胞間質化的影響及機制之探討
指導教授：	莊永仁 Chuang, Yung Jen
口試委員:	王文德吳長益
學位類別：	碩士 Master
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：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	36
中文關鍵詞：	斑馬魚、心外膜、間質化
外文關鍵詞：	Zebrafish, Epicardium, Epithelial-to-mesenchymal transition
相關次數：	點閱：2 下載：0
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心血管疾病的高致死率，對人類的健康造成極大威脅，在已開發國家中造成的死亡人數比率甚至高於癌症。心肌梗塞的發生，主要是因為血栓在冠狀動脈中堆積使得血管的堵塞，進而導致下游心肌細胞無法藉由血液獲取養分及氧氣而壞死。雖然在哺乳動物上已被證實心肌確實具有微弱的細胞再生能力，但由於再生速度非常緩慢而無法完整修復心臟之功能，最終導致心衰竭而死亡。
相較於哺乳類，斑馬魚在多種器官上擁有非常強大的修復能力，其中也包含心臟。儘管將斑馬魚心臟裁切掉原有的五分之一，經過約莫一個月後，受損部位即可再生回來且功能完好如初，因此斑馬魚在目前再生醫學領域中成為一個非常重要的研究工具。在早期文獻中指出，在斑馬魚心臟損傷初期，心外膜會開始增生並將傷口區域包裹起來，並且表現一些只在發育階段時活化的轉錄調控因子，進而使心外膜細胞進行間質化，藉由這過程，心外膜細胞會進行去分化，再轉變成具有分化能力的前驅細胞，而這些前驅細胞已被證實在心臟修復過程中，能夠分化成多種構成心臟的必要單元，例如: 血管內皮層細胞、內皮層細胞、纖維母細胞…等，進而對受損部位進行修復。
從傷口修復的基本步驟來看，過程包含凝血、發炎反應、組織新生、組織重塑…等重要過程，彼此都有部份時序重疊且互相影響；根據先前的研究成果已證實發炎反應對於斑馬魚進行後續心臟修復過程來說是非常重要的啟動步驟，但發炎反應又是如何去啟動下一階段的修復過程，也就是心外膜細胞的間質化，至今仍未被釐清，因此本篇研究主題是要找出能夠連接發炎反應以及心外膜細胞間質化的關鍵調控分子。實驗結果顯示，前列腺素PGE2可能是此關鍵調控分子，它能夠藉由轉化生長因子-β(TGF-β)訊息傳遞去促使心外膜細胞進行間質化；此外，在活體實驗上也發現到在受傷初期轉化生長因子-β訊息傳遞能夠促使心外膜細胞的活化。這些結果可讓我們釐清生物體在修復受損心臟的過程中所參與的機制以及關鍵性分子，進而應用在未來人類心臟修復的療法以及相關藥物之開發。

Myocardial infarction (MI) occurs when cardiac circulation is disrupted by coagulation, and the affected myocardium was damaged due to insufficient supply of blood. Failure to repair and regenerate damaged cardiac tissues after MI is a major cause of heart failure in human. In contrast, zebrafish heart has an extraordinary regenerative capacity. During zebrafish heart regeneration, the epicardium is activated and undergoes epithelial-to-mesenchymal transition (EMT) to generate epicardium-derived cells (EPDCs). EPDCs then migrate into the injured area where they generate various cell types to replace the damaged tissues.
From our previous studies, it has been demonstrated that inflammation response is critical for triggering the regeneration process in zebrafish heart post injury. However, how the inflammation acts to activate the subsequent regenerative event, namely EMT of epicardium, is still unclear. To gain insight on this question, we targeted the COX-2/PGE2 pathway and aimed to verify whether PGE2 is the key mediator to connect inflammation with EMT during heart regeneration. For this study, we adapted an in vitro cell-based model of zebrafish epicardial cells to accompany in vivo assay as the research system. The epicardial primary cells had an epithelium-like morphology and markedly expressed the cell surface marker tight junction protein 1 (ZO-1). After PGE2 treatment, epicardial cells changed into a spindle-shaped morphology and lost the expression of ZO-1, which is consistent with the known characters of EMT. Interestingly, the expression of ZO-1 was partial restored when we treated the cells with a chemical TGF-β inhibitor SB-431542 (iALK5), which was further verified in vivo. In summary, we concluded that PGE2 is an epithelial-mesenchymal-transition mediator, whose function is mediated through the induction of TGF-β signaling pathway during zebrafish heart regeneration.

ABSTRACT ........................................................................................................................................I
中文摘要.........................................................................................................................................III
ABBREVIATIONS .............................................................................................................................V
CHAPTER 1 – GENERAL INTRODUCTION .......................................................................1
1.1 – Cardiovascular disease ...................................................................................................1
1.2 – Zebrafish as a popular model organism .........................................................................1
1.3 – Heart regeneration in zebrafish .....................................................................................2
1.4 – Epicardial EMT during heart regeneration .....................................................................3
1.5 – The specific aim of this study …………………......................................................................4
CHAPTER 2 – MATERIALS AND METHODS ...................................................................6
2.1 – Zebrafish husbandry & Ethics statement .......................................................................6
2.2 – Ventricular resection & drug delivery for suppressing TGF-β signaling ..........................6
2.3 – RNA isolation and RT-Q PCR (real-time quantitative PCR) .............................................7
2.4 – Histology ........................................................................................................................8
2.5 – Immunofluorescence staining ........................................................................................9
2.6 – Primary epicardium cell explants assay ..........................................................................9
2.7 – Immunocytochemistry .................................................................................................10
CHAPTER 3 – Epithelial-to-mesenchymal transition in zebrafish epicardium during heart regeneration is regulated by PGE2 through a TGF-β-dependent mechanism …..………...……………………………………………………………….......12
Results .......................................................................................................................................12
3.1 – Establishment of in vitro zebrafish epcardial cells culture ...........................................12
3.2 – PGE2 induced EMT in primary epicardial cells ………………………………………………............12
3.3 – PGE2 induced EMT through a TGFβ dependent mechanism .......................................13
3.4 – Inhibition of TGFβ/Activin signaling attenuates epicardium activation at the early state of zebrafish heart regeneration ………………………………………………………………………………….......15
CHAPTER 4 – Conclusion & Discussion ………………………………..…………………………........18
4.1 – The role of PGE2 in the zebrafish epicardial EMT …………………..…………………………….....18
4.2 – The role of TGFβ in fibrous tissue deposition in the healing infarct ............................19
4.3 – TGFβ as a therapeutic target in the infarcted heart .....................................................20
REFERENCES ..................................................................................................................................22
TABLES ...........................................................................................................................................25
Table.1 Primer list for RT-qPCR .............................................................................................25
FIGURES .........................................................................................................................................26
Fig. 1 Primary epicardial cells explant & culture system .......................................................26
Fig. 2 PGE2 induce EMT-like morphological change in zebrafish epicardial cells .................27
Fig. 3 PGE2 can induce EMT in zebrafish epicardial cells ......................................................28
Fig. 4 The functions of TGFβ-signaling pathway in zebrafish epicardial-EMT .......................29
Fig. 5 PGE2 induce epicardial-EMT through a TGFβ/Activin dependent mechanism ...........30
Fig. 6 The activation of epicardium during zebrafish heart regeneration ............................31
Fig. 7 Inhibition of TGFβ/Activin signaling attenuates epicardium activation .......................32
Fig. 8 Time scale and important stages during wound healing .............................................33
Fig. 9 The process of the epicardial-EMT during zebrafish heart regeneration ....................34
APPENDIX FIGURES .......................................................................................................................35
Appendix Fig. 1 The gene expression of COX2 and TGFβ during zebrafish heart repair .......35


                                

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