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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊鎧鍵(Kai-Chien Yang) | |
dc.contributor.author | Chao-Ling Chen | en |
dc.contributor.author | 陳昭伶 | zh_TW |
dc.date.accessioned | 2021-06-08T01:44:33Z | - |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-16 | |
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Petrov, Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts. Methods Find Exp Clin Pharmacol, 2002. 24(6): p. 333-44. 48. Dickhout, J.G., R.E. Carlisle, and R.C. Austin, Interrelationship between cardiac hypertrophy, heart failure, and chronic kidney disease: endoplasmic reticulum stress as a mediator of pathogenesis. Circ Res, 2011. 108(5): p. 629-42. 49. Ishida, Y. and K. Nagata, Hsp47 as a collagen-specific molecular chaperone. Methods Enzymol, 2011. 499: p. 167-82. 50. Wang, J., R. Zohar, and C.A. McCulloch, Multiple roles of alpha-smooth muscle actin in mechanotransduction. Exp Cell Res, 2006. 312(3): p. 205-14. 51. The profile of TXNDC5 protein expression. THE HUMAN PROTEIN ALTALS. http://www.proteinatlas.org/ENSG00000239264-TXNDC5/tissue/primary+data (assecced on 1 Aug 2016). 52. Minamino, T., I. Komuro, and M. Kitakaze, Endoplasmic reticulum stress as a therapeutic target in cardiovascular disease. Circ Res, 2010. 107(9): p. 1071-82. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19084 | - |
dc.description.abstract | 心臟衰竭 (heart failure) 是心臟因為先天異常或後天受損傷造成結構或功能異常,使得心臟搏出量不足以支應身體需求而產生症狀。心臟纖維化(cardiac fibrosis)在心臟衰竭病態之組織重組(remodeling) 扮演重要的一環。心臟纖維化的細胞病理特徵伴隨著心臟纖維母細胞被活化、其活化之分化態肌纖維母細胞(myofibroblast) 形成過程中促使細胞外組織間質膠元蛋白 (collagen)合成與沉積。心臟纖維化過程中產生大量的細胞外組織間質 (extracellular matrix) 堆積進而取代損壞心肌細胞,導致心室舒張功能異常、心肌電氣傳導受阻,增加嚴重心律不整發生的危險性。因此,針對心肌纖維化的治療可以減緩或防止心臟衰竭惡化。然而,目前可選擇的治療藥物相當有限,因此本研究希望找到與新穎的心臟纖維化分子機轉。本研究利用次世代高速核酸定序(Next-generation sequencing)針對人體左心室組織的RNA 表現進行分析,我們發現內質網蛋白TXNDC5 (Thioredoxin domain containing 5, 硫氧還蛋白5) 在心臟纖維化中扮演一個重要的樞紐基因。在人類與小鼠心臟衰竭組織與正常組織相較之下,TXNDC5 在心衰竭組織中具高度表現,其與纖維化相關基因Col1A1, ELN, CTGF 與ACTA2 表現呈現高度相關性,此外,TXNDC5 在小鼠心室也具有組織特異性,主要表現在心室纖維母細胞(ventricular cardiac fibroblasts)。然而,目前TXNDC5 在心臟纖維化過程中的機轉仍然不清楚。
因此我們的研究目標想要探討 (1) TXNDC5 參與心臟纖維化過程中的機轉,包括心臟纖維母細胞活性及基因/蛋白表現的分子機轉;(2) 並在動物模式中探討TXNDC5 在心臟纖維化的功能;(3) 以及未來針對TXNDC5 發展治療心臟纖維化藥物的可能性。 TXNDC5 已知為位於內質網具有雙硫鍵異構酶 (protein disulfide isomerase,PDI),我們發現TXNDC5 於人類與小鼠的心室纖維母細胞在接受促進纖維化因子如 乙型腫瘤生長激素(tumor growth factor-β1, TGF-β1)刺激時,其表現量與心臟衰竭時表現相似。敲弱 (knockdown) 人類與小鼠心室纖維母細胞的TXNDC5 基因之後,我們發現其會減少為TGF-β1 刺激引起纖維化相關蛋白之製造與摺疊(folding),但卻對纖維化相關基因表現沒有影響。另外我們也發現TXNDC5 基因敲弱的人類與小鼠心室纖維母細胞會透過內質網相關蛋白質降解路徑(endoplasmic reticulum associated protein degradation, ERAD) 的蛋白體酶(proteasome) 來降解未折疊之纖維化蛋白。而且TXNDC5 基因的表現為透過TGF-β1 引起內質網壓力 (ER stress) 下游所調控,其會受到ATF6 轉錄因子(Activating transcription factor 6)的調節,進而增加心臟纖維化蛋白質的疊疊與製造。在小鼠動物模型中,我們發現Txndc5-/-小鼠會減少因病理性刺激 (isoproterenol)導致的心臟肥厚、心肌纖維化以及心臟衰竭,改善左心室射出分率 (left ventricular ejection fraction),並減少與纖維化相關蛋白質Col1A1, ELN, CTGF 與ACTA2 的表現。 因此本研究發現心臟組織/細胞受到病理壓力或TGF-β1 刺激,誘發內質網壓力調控下游的轉錄因子活化TXNDC5, TXNDC5 會藉由調控細胞外間質蛋白的折疊與蛋白質降解路徑,影響左心室纖維母細胞的活性與細胞外間質蛋白質的製造,造成心臟纖維化,為程中重要的調節因子。未來我們將設計可專一針對TXNDC5蛋白結合的核酸治療藥物DNA aptamer,發展出治療心臟纖維化及心衰竭的新治療模式與標的。 | zh_TW |
dc.description.abstract | Background:Heart failure (HF), one of the leading causes of increased morbidity, mortality, and healthcare burden, attributed largely to cardiac structural or function abnormalities in response to cardiac injury. Fibrosis plays a pivotal role in pathological remodeling of end-stage organ impairment. Cardiac fibrosis is characterized by the hyperproliferation of cardiac fibroblasts (CF), differentiation of CF into myofibroblasts, and excessive extracellular matrix (ECM) synthesis and deposition. Cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current diagnostic and treatment options for cardiac fibrosis, however, are very limited. Combining RNA sequencing in human failing heart, Weighted Gene Co-expression Network Analysis (WGCNA) and in vitro experiments, we have identified thioredoxin domain containing 5 (TXNDC5), an ER-resident protein, as a potential novel modulator of cardiac fibrosis. TXNDC5 is upregulated in failing human and mouse heart and its expression is highly correlated with the expression levels of fibrosis/ ECM genes including Col1A1, ELN, CTGF and ACTA2. However, it remains unclear how TXNDC5 regulation contributes to cardiovascular pathology such as cardiac fibrosis and heart failure.
Aim: The goal of this study was to investigate: (1) the molecular mechanisms of TXNDC5 modulated cardiac fibrosis; (2) in vivo functional contribution of TXNDC5 to cardiac fibrosis; (3) the therapeutic potential of targeting Txndc5 in preventing or reversing cardiac fibrosis. Results: The ER-resident protein TXNDC5 is highly upregulated in human and mouse failing myocardium and in fibroblasts upon TGF-β1 stimulation. Knocking down TXNDC5 in cardiac fibroblasts abrogates TGF-β1-induced fibroblast activation, and ECM protein but not mRNA upregulation. Further experiments confirmed that TXNDC5 functions by facilitating the folding of ECM proteins; depletion of TXNDC5 leads to ECM protein misfolding and degradation through ER-associated degradation (ERAD) pathway. TGF-β1-induced TXNDC5 expression is dependent on transcriptional regulation downstream of ER stress pathway. To investigate the in vivo functional role of TXNDC5, we have generated Txndc5 KO (Txndc5-/-) mice using CRISPR/Cas9 genome editing technology. Comparing to WT animals, Txndc5-/- mice have reduced cardiac fibrosis/hypertrophy and preserved cardiac function in response to isoproterenol-induced cardiac injury. Furthermore, we tested the hypothesis that pharmacological inhibition of TXNDC5 using disulfide isomerase inhibitor 16F16 and DNA aptamers could be a novel therapeutic approach against cardiac fibrosis and heart failure. Conclusion: We have identified an ER resident protein TXNDC5 as a novel mediator of cardiac fibrosis; Txndc5 regulates fibroblast activity and ECM production by modulating ECM protein folding and ER-associated protein degradation. TGF-β1, a critical mediator of cardiac fibrosis, triggers TXNDC5 expression in an ER stress-dependent manner. Genetic deletion of TXNDC5 protects against β agonist-induced cardiac fibrosis and LV dysfunction. Therefore, TXNDC5 represents a novel molecular mechanism contributing to cardiac fibrosis and could be a novel therapeutic target against cardiac fibrosis and heart failure. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:44:33Z (GMT). No. of bitstreams: 1 ntu-105-R03443001-1.pdf: 6200134 bytes, checksum: 38f76466beecfc0420daf9e7146aa53a (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目 錄
致謝…………………………………………………………………………………………I 中文摘要………………………………………………………………………………II 英文摘要………………………………………………………………………………VI 目錄………………………………………………………………………………………VII 圖目錄…………………………………………………………………………………X 表目錄…………………………………………………………………………………XI 縮寫表…………………………………………………………………………………XII Chapter 1 Introduction……………………………………………………………………………… 1 1.1 Heart failure and cardiac fibrosis …………………………………… 1 1.2 Cardiac fibroblast and fibroblast activation ………… 3 1.3 Endoplasmic reticulum (ER) stress ……….…………………………. 3 1.3.1 UPR (unfolded protein response)……………………… 3 1.3.2 PERK (PKR-like ER kinase)……………………………………… 4 1.3.3 IRE-1 (inositol-requiring enzyme 1)………………4 1.3.4 ATF6 (activating transcription factor)………5 1.3.5 ER-associated protein degradation (ERAD)…6 1.4 Thioredoxin domain containing protein 5(TXNDC5)…7 1.4.1 TXNDC5 protein structure…………………………………………………7 1.4.2 TXNDC5 protein functions…………………………………………………8 1.4.3 TXNDC5 transcriptional regulation…………………………8 1.5 Aim of study……………………………………………………………………9 Chapter 2 Material and methods………………………………………………………………10 2.1 Generation of TXNDC5 knockout (KO) mice using CRISPR/Cas9 genome-editing technology………………………………………………10 2.2 Isoproterenol (ISO)-induced cardiac fibrosis…………………11 2.3 Echocardiography……………………………………………………………………………………………11 2.4 Histology…………………………………………………………………………………………………………….12 2.5 Immunohistochemistry…………………………………………………………………………………12 2.6 Picrosirius red staining………………………………………………………………………13 2.7 Primary mouse cardiac fibroblasts isolation and culture……………………… 14 2.8 Isolated human ventricular myocyte samples………………………14 2.9 Human ventricular fibroblast (HuVF) cultures…………………15 2.10 Cell line and primary mouse cardiac fibroblasts culture………………………………………………………………………………………………………………………………17 2.11 Lentiviral transduction……………………………………………………………………………………………………………….17 2.12 RNA extraction and qRT-PCR…………….………………………………………………18 2.13 Western blot analysis……….…….……………………………………………………….18 2.14 Immunofluorescent staining for fixed cells and tissues………………………………………………………………………………………………………………………………19 2.15 Collagen measurement………………………………………………………………………………20 2.16 Fibroblast proliferation assay…………….………………………………….21 2.17 Protein stability assay……….…….………………………………………………….22 2.18 Proteasome inhibition assay……………………………………………………………22 2.19 ER stress inhibition assay………………………………………………………………22 2.20 Promoter luciferase assay……….…………………………………………………….23 2.21 FRET (fluorescence resonance energy transfer) based protein folding assay.…………………………………………………………………………………….23 Chapter 3 Results……………………………………………………………………………………………………25 3.1 TXNDC5 is highly expressed in failing human heart……25 3.2 TXNDC5 is induced by isoproterenol stimulation in mouse heart …………………………………………………………………………………………………………………26 3.3 Knocking down TXNDC5 in cardiac fibroblasts abrogates TGF-β1-induced fibroblast activation and ECM protein, but not mRNA, upregulation.……………………………………………………………………………………27 3.4 TXNDC5 depletion accelerates ECM protein degradation in a proteasomen Dependent manner………………………………………………………28 3.5 TXNDC5 facilitates ECM protein folding in endoplasmic reticulum ………………………………………………………………………………………………………………………30 3.6 TXNDC5 is regulated by TGF-β1-ER stress pathway ….…………………………………………………………………………………………………………………………………………………31 3.7 An ER stress element ATF6 is responsible for TGF-β1-induced TXNDC5 transcriptional regulation ….……………….…………31 3.8 The in vivo role of TXNDC5 in modulating cardiac fibrosis….…………………………………………………………………………………………………………………………………………………32 Chapter 4 Discussions ……………………………………………………………………………………34 4.1 Mechanisms of cardiac fibrosis: the role of TGFβ pathway……….……………………………………………………………………………………………………………………34 4.2 The physiological and pathological TGFβ1 concentration in cardiac fibroblasts…………………………………………….35 4.3 The role of ER stress and unfolded protein response (UPR) in cardiac fibrosis. …………………………………………………………………….36 4.4 Genetic delection of TXNDC5 does not affect normal development…………………………………………………………………………………………………………………38 4.5 TXNDC5 as a critical regulator and potential therapeutic target of cardiac fibrosis…………………………………39 4.6 DNA aptamer targeting TXNDC5 as a potential therapeutic agent against heart failure and cardiac fibrosis………….………………………………………………………………………………………………………..41 References. …………………………………………………………………………………………………………….42 Figures and tables . ………………………………………………………………………………………46 Appendix…………………………………………………………………………………………………………………………72 圖目錄 FigureⅠ. The physiopathology of cardiac fibrosis ……………2 FigureⅡ. Schematic figure to illustrate ER stress pathway and UPR…………………………………………………………………………………………………………5 FigureⅢ. Endoplasmic-reticulum-associated protein degradation (ERAD) pathways……………………………………………………………………………6 FigureⅣ. Schematic structure of TXNDC5 domains….………………7 FigureⅤ. Schematic diagram of interaction among profibrotic TGF-β signaling pathways…………………………………………………35 FigureⅥ. The mechanism of TXNDC5 in cardiac fibrosis…37 FigureⅦ. The profile of TXNDC5 protein expression in cardiomyocytes and smooth muscle cells……………………………………………38 Figure 1. TXNDC5 is highly expressed in failing heart in human.………………………………………………………………………………………………………………………………………………46 Figure 2. TXNDC5 is essential in ISO-induced cardiac fibrosis..………………………………………………………………………………………………………………………50 Figure 3. TXNDC5 modulates ECM expression on the protein, instead of mRNA, level in human cardiac fibroblasts (HCF). ……………………………………………………………………………………………53 Figure 4. TXNDC5 modulates ECM expression on the protein, instead of mRNA, level in mouse cardiac fibroblasts. ………………………………………………………………………………………………………..56 Figure 5. Silencing of TXNDC controls ECM protein stability through ERAD pathway…………………………………………………..…………59 Figure 6. FRET (fluorescence resonance energy transfer)-based protein protein assay……………………………………………………………….…..61 Figure 7. TXNDC5 is controlled by TGFβ and ER stress pathway.…………………………………………………………………………………………………………………………………………… 63 Figure 8. ATF6 is the regulation of Txndc5 promote activity.……………………………………………………………………………………………65 Figure 9. TXNDC5-/- mouse was generated by CRISPR genomic editing method…68 Figure 10. Txndc5 knockout reduced cardiac fibrosis and preserved cardiac functio…69 Figure S1: pGL3-mTXNDC5 (-1000~+1000)……..…………..………………….74 Figure S2: pGL3-mTXNDC5 -1000~+1000 (+769~+780 deleted) ………………74 Figure S3: pEBFP-EGFP……..…………..…………….……..…………..………75 Figure S4: pEBFP-IRES-EGFP……..…………..…………….……..…………..…75 Figure S5: pEBFP-mouse Col1A1-EGFP……..…………..……………….……..76 Figure S6: pEBFP-mouse Eln-EGFP……..…………..…………….……..………76 表目錄 Tabel 1. Drugs affecting UPR and ER-initiated in cardiovascular diseases………40 Table S1: Mouse primer sequence used in this study………… 72 Table S2: Human primer sequence used in this study……………73 | |
dc.language.iso | en | |
dc.title | 內質網蛋白TXNDC5(硫氧還蛋白5)在心臟纖維化與心臟衰竭的角色 | zh_TW |
dc.title | The Role of ER-Resident Protein TXNDC5 in Cardiac Fibrosis and Heart failure | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林水龍(Shuei-Liong Lin),陳青周(Ching-Chow Chen),陳文彬(Wen-Pin Chen) | |
dc.subject.keyword | 心臟纖維化,心臟衰竭,TXNDC5(硫氧還蛋白5),內質網壓力, | zh_TW |
dc.subject.keyword | cardiac fibrosis,thioredoxin domain containing 5,TXNDC5,endoplasmic reticulum stress, | en |
dc.relation.page | 76 | |
dc.identifier.doi | 10.6342/NTU201602844 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-16 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥理學研究所 | zh_TW |
顯示於系所單位: | 藥理學科所 |
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