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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80447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 | Chih-Fan Yeh | en |
| dc.contributor.author | 葉志凡 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:06:51Z | - |
| dc.date.available | 2021-12-30 | |
| dc.date.available | 2022-11-24T03:06:51Z | - |
| dc.date.copyright | 2021-12-30 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-12-09 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80447 | - |
| dc.description.abstract | 基本原理 動脈彎曲和分叉處的擾流會激活內皮細胞，進而造成動脈粥狀硬化。目前動脈粥狀硬化的藥物治療主要是針對風險因素的控制，例如糖尿病、高血壓、高血脂等的慢性疾病的控制跟治療，而非血管本身。此外，這些治療對於動脈粥狀硬化的改善仍不盡理想。因此，這樣的缺口，也凸顯了釐清和靶向內皮中引起動脈粥狀硬化的新型機械敏感性機制的重要性。 實驗目標 探討內質網蛋白Thioredoxin Domain Containing 5 (TXNDC5)在動脈粥狀硬化致病過程中的角色，釐清TXNDC5於內皮機械傳導路徑中的分子機制，並針對此機制結合奈米醫學的技術去剔除內皮中的TXNDC5，以達成治療動脈粥狀硬化的目標。 實驗方法和結果 TXNDC5分別在人類及小鼠的動脈粥狀硬化病灶中，被發現其蛋白表現量顯著增加。首先，我們利用三種新型小鼠疾病模式來證明TXNDC5高度表達於受擾流刺激下的血管內皮細胞中。再者，針對性地剔除小鼠內皮細胞中的TXNDC5可顯著的減少動脈粥狀硬化的發生。機制上，我們闡明內皮細胞中的TXNDC5 透過破壞eNOS蛋白的穩定性，導致內皮功能的失常，進而造成動脈粥狀硬化的發生。TXNDC5 可增加熱休克反應（heat shock response）中重要的轉錄因子HSF1的泛素化（ubiquitination）以及蛋白酶體(proteasome)媒介的降解，導致其下游蛋白HSP90表現量減少，最終致使eNOS蛋白的穩定性下降。為了要證實TXNDC5是可作為治療動脈粥狀硬化的標的，我們整合奈米微粒及內皮特異性啟動子 (CDH5) 驅動的 CRISPR/Cas9 系統，來標靶性剔除內皮細胞中的Txndc5，結果證實此策略顯著增加eNOS蛋白的表現並減少ApoE-/-小鼠動脈粥狀硬化的發生。 結論 本研究藉由細胞和動物實驗，闡述血液擾流誘導的內皮TXNDC5 在動脈粥狀硬化中的詳細機轉。此外，我們藉由CRISPR技術設計一個奈米醫學平台，在體內有效且特異性地剔除內皮細胞中的Txndc5，從而減少動脈粥狀硬化發生，以此建立一個經由靶向內皮機械敏感的途徑作為治療動脈粥狀硬化的概念，並期望其作為未來的治療方針。 | zh_TW |
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| dc.description.tableofcontents | "誌謝 1 中文摘要 3 ABSTRACT 5 CONTENTS 7 LIST OF FIGURES 10 Chapter 1 Introduction 12 1.1 Atherosclerosis 12 1.1.1 Epidemiology of atherosclerotic cardiovascular disease 12 1.1.2 Mechanism of atherosclerosis 12 1.1.3 Contribution of hemodynamic flow on atherosclerosis 14 1.1.4 Functions of flow-sensitive endothelial nitric oxide synthase (eNOS) 15 1.2 Thioredoxin domain containing 5 (TXNDC5) 16 1.2.1 PDI function of TXNDC5 16 1.2.2 TXNDC5 and human diseases 16 1.2.3 TXNDC5 in endothelial cells 17 1.3 Aim of the study 17 Chapter 2 Material and Methods 18 2.1 Generation of Txndc5-/- mice, Txndc5fl/fl mice, and cell type-specific Txndc5 conditional knockout mice 18 2.2 Generation of cell type-specific fluorescence reporter mice 20 2.3 Partial carotid artery ligation (PCAL) 20 2.4 AAV9-PCSK9 mouse model 21 2.5 Intimal RNA isolation from carotid arteries 21 2.6 Plaque lesion analysis of aorta, aortic sinuses and carotid arteries 21 2.7 Immunofluorescence staining 22 2.8 En face staining 23 2.9 Human aortic endothelial cell (HAEC) culture 23 2.10 Athero-relevant cone plate flow system 23 2.11 siRNA transfection of HAEC 23 2.12 Lentiviral transduction of HAEC 24 2.13 mRNA transfection of HAEC 24 2.14 RNA extraction and qRT-PCR 25 2.15 Immunoblot analysis 25 2.16 Cycloheximide protein stability assay 26 2.17 Co-immunoprecipitation for TXNDC5-interacting proteins and ubiquitination of HSF1 26 2.18 TXNDC5 promoter luciferase reporter assay 26 2.19 Nitric oxide production assay 27 2.20 Transendothelial electrical resistance 27 2.21 RNA sequencing and pathway enrichment analysis 27 2.22 Re-analysis of publicly available microarray datasets 28 2.23 Statistical analysis 28 2.24 Graphical abstract creation 28 Chapter 3 Results 29 3.1 TXNDC5 is induced in the endothelium exposed to DF and upregulated in atherosclerotic lesions 29 3.2 Global and endothelium-specific deletion of Txndc5 significantly reduces atherosclerosis in vivo 31 3.3 Disturbed flow-induced TXNDC5 downregulates eNOS protein in vascular endothelium 33 3.4 TXNDC5 destabilizes eNOS protein by reducing HSP90 in endothelial cells 35 3.5 TXNDC5 suppresses HSP90 expression via ubiquitin-dependent HSF1 degradation in the endothelium 36 3.6 Endothelial TXNDC5 is transcriptionally repressed by flow-sensitive transcription factor KLF2 38 3.7 Endothelial Txndc5 deletion achieved by a targeted nanomedicine platform significantly reduces atherosclerosis in ApoE-/- mice 39 Chapter 4 Discussion 41 4.1 Mechano-sensitive endothelial TXNDC5 is a novel target for atherosclerosis 41 4.2 The novel mechanism by which TXNDC5 regulates eNOS protein stability 41 4.3 Novel link of heat shock factor 1 to atherosclerosis via mechano-sensitive TXNDC5 42 4.4 Advantage of targeting TXNDC5 to treat atherosclerosis 43 4.5 The advantage of nanoparticle-based treatment targeting vascular endothelium 45 REFERENCE 47 FIGURES 53 Figure 1. Generation of Txndc5fl/fl mice using CRISPR/Cas9-based genome editing. 53 Figure 2. Experimental scheme of partial carotid ligation and AAV9-PCSK9 atherogenic models. 54 Figure 3. TXNDC5 is induced in the endothelium exposed to disturbed flow. 55 Figure 4. TXNDC5 expression is significantly upregulated in endothelial cells exposed to disturbed flow and in atherosclerotic lesions. 58 Figure 5. Global deletion of disturbed flow-induced Txndc5 significantly reduces atherosclerosis in vivo. 61 Figure 6. No change of plasma lipid profile or inflammatory markers by Txndc5 deletion in ApoE-/- mice. 62 Figure 7. Endothelial deletion of disturbed flow-induced Txndc5 significantly reduces atherosclerosis in vivo. 64 Figure 8. Disturbed flow upregulates endothelial TXNDC5 to increase protein degradation of endothelial nitric oxide synthase (eNOS). 68 Figure 9. TXNDC5-mediated endothelial dysfunction and atherosclerosis are eNOS/nitric oxide-dependent. 70 Figure 10. TXNDC5 downregulates HSP90 to destabilize eNOS protein in endothelial cells. 73 Figure 11. RNA sequencing analysis identifies heat shock response regulated by TXNDC5 depletion. 74 Figure 12. TXNDC5 downregulates eNOS by transcriptional regulation of HSP90. 76 Figure 13. TXNDC5 downregulates HSF1 protein expression without affecting its transcripts. 77 Figure 14. TXNDC5 increases ubiquitin-dependent HSF1 degradation to decrease HSP90 and eNOS in vascular endothelium. 80 Figure 15. Efficiency of KLF2 overexpression and its binding sites at TXNDC5 promoter region. 81 Figure 16. Endothelial TXNDC5 is transcriptionally suppressed by KLF2. 82 Figure 17. In vivo administration of nanoparticles carrying an endothelium-specific Txndc5-targeting CRISPR/Cas9 vecteor deletes endothelial TXNDC5 and ameliorates disturbed flow-induced carotid atherosclerosis. 84 Figure 18. Proposed athero-relevant mechano-transduction mechanisms mediated by TXNDC5 in vascular endothelium. 85 Table 1. Primer list 86 Table 2. Antibody list 88 Table 3. List of dysregulated genes in TXNDC5-depleted HAEC exposed to disturbed flow. 89" | |
| dc.language.iso | en | |
| dc.subject | 奈米醫學 | zh_TW |
| dc.subject | 內皮型一氧化氮合成酶 | zh_TW |
| dc.subject | 動脈硬化 | zh_TW |
| dc.subject | 剪應力 | zh_TW |
| dc.subject | 內皮細胞 | zh_TW |
| dc.subject | TXNDC5 | en |
| dc.subject | Atherosclerosis | en |
| dc.subject | endothelial cell | en |
| dc.subject | shear stress | en |
| dc.subject | nanomedicine | en |
| dc.subject | endothelial nitric oxide synthase | en |
| dc.title | 透過靶向血管內皮細胞的機械敏感性蛋白TXNDC5以穩定eNOS並改善動脈粥狀硬化 | zh_TW |
| dc.title | Targeting mechano-sensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.author-orcid | 0000-0002-0678-2264 | |
| dc.contributor.advisor-orcid | 楊鎧鍵(0000-0002-9128-9166) | |
| dc.contributor.oralexamcommittee | 高憲立(Hsin-Tsai Liu),裘正健(Chih-Yang Tseng),黃柏勳,劉秉彥,陳文彬 | |
| dc.subject.keyword | 動脈硬化,剪應力,內皮細胞,內皮型一氧化氮合成酶,奈米醫學, | zh_TW |
| dc.subject.keyword | Atherosclerosis,TXNDC5,shear stress,endothelial cell,endothelial nitric oxide synthase,nanomedicine, | en |
| dc.relation.page | 92 | |
| dc.identifier.doi | 10.6342/NTU202104521 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-12-09 | |
| 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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