斑馬魚心臟修復早期過程中Syndecan-4的功能｜國立清華大學博碩士論文庫

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研究生：	賴亭羽 Lai, Ting-Yu
論文名稱：	斑馬魚心臟修復早期過程中Syndecan-4的功能 The characterization of syndecan-4 in early stage of adult zebrafish heart regeneration
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:	鄭世進 Cheng, Shih-Chin 陳振輝 Chen, Chen-Hui
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：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	42
中文關鍵詞：	斑馬魚、冷凍創傷、心臟再生、Syndecan-4 、心電圖
外文關鍵詞：	Zebrafish, Cryoinjury, Heart regeneration, Syndecan-4, Electrocardiogram
相關次數：	點閱：2 下載：0
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心肌梗塞後的組織修復牽涉到複雜的機制，而這些修復的過程中，發炎反應被猜測是關鍵的初始步驟。缺氧壞死的組織誘發細胞介素和趨化介素等因子之表現，調控先天性免疫反應的同時，影響各式生長因子，進而調控血管新生、心肌細胞再生、以及組織重塑等後續反應。大多數哺乳類動物的心臟受損後，再生與組織重塑被認為會互相抗衡，導致受傷的心肌組織由纖維化瘢疤所取代。而這些不具彈性的纖維化組織，會對正常收縮的心肌造成傷害，嚴重時可能造成心臟衰竭而死亡。相較而言，斑馬魚在心肌受損後，具有無疤再生的修復能力，這意味著透過了解斑馬魚心臟再生過程中的調控機制，有助於我們釐清有那些因子參與調控受損心臟傷疤的生成與退減。
先前在脊椎動物的研究顯示，syndecan-4 (Sdc4)可以調節發炎反應和纖維母細胞的活性。有趣的是，有研究指出剔除Sdc4的小鼠在心肌梗塞後心臟較容易破裂。因此，我們猜測在斑馬魚心臟創傷初期，Sdc4可能透過調控發炎反應及細胞外基質生成，藉以維持心臟功能完整，同時也影響了心臟再生修復的進程。為了驗證這一個假設，我們先進行斑馬魚心臟創傷後的基因表現時序分析。結果顯示，創傷後初期sdc4的表現量果然快速上升，暗示其重要性。接著透過注射接附sdc4 siRNA的奈米磁珠，達成抑制斑馬魚心臟內sdc4基因表現，以進行後續實驗。我們利用轉殖螢光魚標定非淋巴球白血球 (myeloid cells)，追蹤白血球聚集於受傷組織的進程。實驗結果顯示減少Sdc4表現可能會阻礙先天免疫細胞的聚集。另外，當sdc4基因被抑制時，col1a1 和tgfb1a的基因表現以及fibronectin蛋白表現量亦隨同下降。透過細胞新生標定結果，我們認為Sdc4可能影響心外膜下層細胞活化與增生。最後，我們透過實驗室先前建立的斑馬魚心電圖測量技術，結合即時錄影，分析心肌收縮狀態與心電圖。由影像可推測sdc4被抑制後，斑馬魚心臟傷口的修復能力下降，以及傷口周邊的心肌組織收縮異常；且同時心電圖中，心肌梗塞指標ST段上升(ST-elevation)持續時間延長。我們假設這些免疫反應、胞外基質調控、以及細胞新生的變化，是抑制sdc4後導致傷口癒合延遲的原因,證實Sdc4在早期心臟修復過程中的重要性。

Damage repair after myocardial infarction involves a series of complex events, which are thought to be initiated by inflammatory responses. The infarcted necrosis tissue induces production of cytokines and chemokines, which recruit immune cells and lead to the generation of several growth factors to induce and regulate angiogenesis, myocardial proliferation, and tissue remodeling.
In most mammals, the disproportion of regeneration and tissue remodeling causes the injured tissue to be replaced by fibrotic scars. These non-elastic fibrosis tissues block the contraction of the heart and damage the normal cardiac muscle tissue, which may result in heart failure and death. In contrast, zebrafish has the ability to undertake scar-free healing after cardiac injury. This implies that if we could understand the mechanism of heart regeneration in zebrafish, we may adapt the knowledge to manage heart repair in human myocardial infarction.
Previous studies have reported that syndecan-4 (Sdc4) regulates the inflammatory response and fibroblast activity in the vertebrates. Interestingly, Sdc4 knockout mice have higher mortality rate after myocardial infarction due to heart wall rupture. Therefore, we hypothesized that in early stage of zebrafish heart repair, Sdc4 may be a key regulator of inflammatory response and the production of extracellular matrix supportive proteins to protect heart from further damage and to promote tissue regeneration. To test this hypothesis, we first conducted a time-lapsed gene expression analysis to identify the gene expression profile of sdc4 after heart injury in zebrafish. As expected, we found the sdc4 expression increased rapidly after cryoinjury, which implies its importance in the early phase of heart regeneration. Then, we injected nano-magnetic beads coupled with sdc4 siRNA to inhibit sdc4 expression in zebrafish heart under an external magnetic field. We used the Lyz:DsRed zebrafish line to label myeloid cells to track the movement of immune cells. The results showed that loss of Sdc4 blocked the accumulation of innate immune cells in the lesion. In addition, when sdc4 was knocked down, we found the gene expression of col1a1a and tgfb1a and the protein expression of fibronectin were all down-regulated. Moreover, loss of Sdc4 might also affect the cell activation and proliferation around subepicardium. Finally, we measured the electrocardiogram in post-heart injury zebrafish with integrated video recording of ex vivo heart. After sdc4 knockdown, we observed the reduced capacity of wound repair and abnormal ventricular contraction around the cryoinjury lesion, meanwhile, the electrocardiogram showed sustained ST-elevation, the marker of myocardial infarction, in sdc4 knockdown group. In conclusion, Sdc4 might mediate the innate immune cells’ movement, the production of ECM proteins, the cell proliferation, the heart’s contractility, and the cardiac repair in the early stage of zebrafish heart repair. Sdc4 could be a critical regulator for scar-free healing in zebrafish heart regeneration.

中文摘要    I
Abstract III
致謝 V
List of Tables VIII
List of Figures VIII
Abbreviations IX
1. Introduction    1
1.1 Current status of myocardial infarction 1
1.1.1 Myocardial infarction 1
1.1.2 Diagnosis and treatment of MI 1
1.1.3 Recent research strategies in MI 3
1.2 Syndecan-4 4
1.2.1 Syndecan-4 is a multi-functional protein 4
1.2.2 Implications of syndecan-4 in heart diseases 5
1.3 Zebrafish model for cardiac tissue repair research 5
1.4 The aim of this study: to investigate the role of syndecan-4 in early stage of zebrafish heart regeneration 6
2. Material and methods 7
2.1 Zebrafish husbandry 7
2.2 Zebrafish anesthetization and sacrifice 7
2.3 Cryoinjury MI model in zebrafish 7
2.4 Adult zebrafish retro-orbital injection 8
2.5 Inducing sdc4 knockdown of the heart in adult zebrafish 8
2.6 RNA isolation and Real-time quantitative PCR analysis 8
2.7 Fixation and cryosection 9
2.8 Immunofluorescence staining 9
3. Results 11
3.1 The SilenceMag sdc4 siRNA inhibited sdc4 gene expression in post-MI heart of adult zebrafish 11
3.2 Sdc4-dependent recruitment of innate immune cells 11
3.3 Sdc4 stimulated the expression of marker genes involved in ECM remodeling 12
3.4 sdc4 regulated the expression of fibronectin in and around the injury site 13
3.5 Dynamic expression of fibronectin was regulated by Sdc4 during cardiac repair 14
3.6 Cell proliferation 15
3.7 Assessing the heart function by electrocardiography and video recording during regeneration 16
3.8 Combined analysis of electrocardiography and video recording on ST-elevation and ventricular aneurysm under sdc4 knockdown 17
4. Conclusion and Discussion 19
4.1 Sdc4 regulates early immune response during heart regeneration 19
4.2 Sdc4-dependent mechanism in heart regeneration 19
4.3 The improved ECG for adult zebrafish 21
5. Reference 22

List of Tables
Table. 1 The zebrafish siRNA sequences 27
Table. 2 Primer list of Real Time-quantitative PCR analysis 27
List of Figures
Figure. 1 Protocol for gene knockdown experiment and the sdc4 gene expression profile in post-injury heart of zebrafish 28
Figure. 2 Sdc4 knockdown reduced the recruitment of innate immune cells in the lesion site following cryoinjury 29
Figure. 3 Target gene expression profiles during cardiac repair 30
Figure. 4 Cryoinjury-induced sdc4 expression co-localized with fibronectin expression around and within the injury site 31
Figure. 5 sdc4 knockdown inhibited fibronectin expression in the lesion zone at 1 dpci 32
Figure. 6 Fibronectin expression in the epicardium and the lesion site was affected by sdc4 knockdwon at 3 dpci 33
Figure. 7 sdc4 knockdown disrupted the cardiac repair process at 7 dpci 34
Figure. 8 Cell proliferation pattern at 3 and 7 dpci 35
Figure. 9 The design of electrocardiography setting and video record 36
Figure. 10 Cryoinjury immediately induces ST-elevation 37
Figure. 11 ST-elevation was persistence under sdc4 knockdown 38
Figure. 12 Video recordings of post-cryoinjury heart 39
Figure. 13 Proposed scheme on the effects of sdc4 knockdown in zebrafish heart repair 40

Figure. S1 Cryoinjury tool and cryoinjury procedure 41
Figure. S2 Inducing sdc4 knockdown of the heart in adult zebrafish. 42


                                

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