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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孟子青(Tzu-Ching Meng) | |
| dc.contributor.author | Shu-Fang Hsu | en |
| dc.contributor.author | 許淑芳 | zh_TW |
| dc.date.accessioned | 2021-05-14T17:43:00Z | - |
| dc.date.available | 2018-08-28 | |
| dc.date.available | 2021-05-14T17:43:00Z | - |
| dc.date.copyright | 2015-08-28 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4526 | - |
| dc.description.abstract | 腫瘤壞死因子alpha (Tumor necrosis factor-alpha, TNF-alpha)是一種多功能性的促進炎症細胞激素，在感染所造成的細胞損傷中對於先天免疫系統(innate immune system)有著重要的調控作用。同時TNF-alpha刺激血管內皮細胞(vascular endothelial cell)會藉由複雜的細胞內信息調控造成細胞發炎反應甚至是動脈粥狀硬化(atherothrombosis)以及發炎性疾病(inflammatory disease)的發生。在發炎反應過程中，TNF-alpha啟動一連串細胞激酶(kinase)的訊息傳遞，活化核轉錄因子(nuclear factor kappa B, NF-kappaB)，促進細胞表面黏著分子(adhesion molecule)的表現以及後續白血球細胞(leukocyte)的附著。在此過程中，人們對於細胞激酶的功能有相對的了解，目前我們並不清楚蛋白質磷酸水解酶(protein phosphatase)是否同樣參與調控TNF-alpha造成的訊息傳遞。在本論文中，我們探討雙專一性磷酸水解酶(dual specificity phosphatases, DUSPs)在TNF-alpha調控內皮細胞發炎反應中的角色扮演。藉由偵測基因表現的mRNA含量，在人類內皮細胞株EAhy926中我們找到一群經TNF-alpha誘導表現的DUSPs。我們也發現TNF-alpha誘導表現的細胞黏著分子(intercellular adhesion molecule-1, ICAM-1)在經微小RNA干擾(RNAi)造成的DUSP6基因剔除實驗中，表現量明顯下降；後續單核球(monocyte)在內皮細胞表面附著的數量也隨之下降，顯示DUSP6在調控發炎反應有正向的作用。我們接著利用人類初代臍靜脈內皮細胞(human umbilical vein endothelial cells, HUVECs)來研究調控機制。結果顯示，在TNF-alpha刺激的HUVEC細胞中，DUSP6藉由抑制細胞外訊號調節激酶(extracellular signaling-regulated kinase, ERK)的活性而促進NF-kappaB的轉錄活性以及其下游ICAM-1的表現。在小鼠的血管組織切片染色(immunohistochemistry, IHC)中，我們也觀察到ICAM-1的表現量在DUSP6基因剔除(Dusp6-/-)小鼠低於野生型小鼠，證實DUSP6確實扮演促進血管內皮發炎的角色。此外，相較於野生型小鼠的敏感，DUSP6基因剔除小鼠對於脂多醣內毒素(lipopolysaccharide, LPS)所造成的敗血性肺部損傷有較佳的抵禦能力。這些結果證實了DUSP6有促進內皮細胞發炎反應及發炎相關病理過程的新穎角色，顯示其作為治療發炎性疾病藥物開發的新契機。 | zh_TW |
| dc.description.abstract | Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that directs multiple events of the innate immune system during infection of cell injury. Meanwhile, TNF-alpha activates a diverse array of signaling pathways in vascular endothelial cells (ECs), leading to the inflammatory phenotype that contributes to the pathogenesis of atherothrombosis and inflammatory diseases. In a typical inflammatory response, TNF-alpha initiates a kinase-dependent signaling cascade, which activates nuclear factor (NF)-kappa B, leading to inducible expression of adhesion molecules and recruitment of leukocytes. In contrast to the known function of kinases in this context, it is not clear whether protein phosphatases participate in the regulation of TNF-alpha signaling. In the present study, we have investigated the role of dual specificity phosphatases (DUSPs) in TNF-alpha-induced inflammatory response. Using human endothelia, EAhy926, for screening of mRNA levels, we identified a group of DUSPs to be inducibly expressed under the TNF-alpha stimulation. Among them, DUSP6 functioned as a prominent positive regulator of the inflammatory response, evidenced by a clear decrease of TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) and a drastic reduction of monocyte adhesion on the surface of endothelia when DUSP6 was ablated via RNAi. We further examined the underlying mechanism controlled by DUSP6 using primary human umbilical vein endothelial cells (HUVECs). Our data showed that inducible DUSP6 promoted canonical NF-kappaB-dependent increase of adhesion molecules exclusively through inhibition of extracellular signaling-regulated kinase (ERK) in TNF-alpha-stimulated human ECs. The role that DUSP6 plays in facilitating endothelial inflammation in aorta and vein was confirmed by in vivo experiments using Dusp6-/- mice. Furthermore, genetic deletion of Dusp6 significantly reduced the susceptibility to inflammatory responses in a mouse model of lung sepsis. These results are the first to demonstrate a novel function of DUSP6 in the regulation of vascular inflammatory response and the underlying mechanism through which DUSP6 promotes endothelial inflammation-mediated pathological process. Our findings suggest that inhibition of DUSP6 holds great potential for the treatment of inflammatory diseases. | en |
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| dc.description.tableofcontents | TABLE OF CONTENTS
誌謝 i 摘要 ii ABSTRACT iii ABBREVIATION iv TABLE OF CONTENTS v LIST OF FIGURES ix LIST OF SCHEMES xii LIST OF TABLES xii CHAPTER 1: INTRODUCTION 1 1.1 The endothelium function and endothelial inflammation 2 1.2 TNF-alpha signaling in regulating endothelial inflammation 3 1.2.1 TNF-alpha induces cell adhesion molecules expression on endothelium 3 1.2.2 TNF-alpha activates canonical NF-kappa B pathway to regulate ICAM-1 expression 5 1.2.3 TNF-alpha-induced MAPKs activation in endothelial inflammation 5 1.3 Role of DUSPs in regulating MAP kinase and cell inflammation 7 1.4 Study the role of DUSPs targeting on ERK to regulate endothelial inflammation 9 CHAPTER 2: MATERIALS AND METHODS 11 2.1 Reagents 12 2.2 Cell culture and transient transfection 12 2.2.1 Culture conditions for each cell line 12 2.2.2 Transient cell transfection 13 2.3 Immunoblotting and antibodies 14 2.4 RNA extraction and quantitative real-time PCR 15 2.5 Monocyte adhesion assay 16 2.6 DUSP6 expression plasmids and luciferase reporter constructs 16 2.7 NF-kappa B reporter assay 17 2.8 RNA extraction and Gene expression profiling 18 2.9 Animal studies 18 2.9.1 Mice housing 18 2.9.2 Genotyping 19 2.9.3 Tail vein injection with TNF-alpha 20 2.9.4 Immunohistochemstry staining and image quantification 20 2.9.5 LPS-induced experimental sepsis and neutronphil adoptive transfer 21 2.9.6 Neutrophil isolation from mouse blood 22 2.9.7 Flow cytometry analysis 22 2.10 Exploring DUSP6-mediated phosphorylation network in TNF-alpha-activated HUVECs by MS analyss 23 2.10.1 Sample preparation for MS/MS analysis 23 2.10.2 In-solution protein digestion 23 2.10.3 TiO2 beads enrichment 24 2.10.4 Immunoprecipitation for phosphotyrsine peptide enrichment 24 2.10.5 Shotgun proteomic identifications 25 2.10.6 Data analysis 26 2.11 Statistical analysis 26 CHAPTER 3: RESULTS 27 3.1 TNF-alpha treatment triggers MAPKs transient activation rather than cell apoptosis in endothelial EAhy926 cells 28 3.2 DUSPs are inducibly expressed in endothelial cell exposed to TNF-alpha and function as MKPs 29 3.3 DUSP6 involves in TNF-alpha-induced endothelial inflammation by regulating intercellular adhesion molecules 1 (ICAM-1) expression 31 3.4 Inducible DUSP6 regulates TNF-alpha-directed inflammatory responses in primary endothelial HUVECs 32 3.5 DUSP6-mediated termination of ERK activity is essential for TNF-alpha-induced inflammatory response in endothelium 34 3.6 Inhibition of ERK by DUSP6 promotes NF-kappa B transcriptional activation in endothelium exposed to TNF-alpha 36 3.7 TNF-alpha-induced ICAM-1expression on the endothelial layer of aorta and vein is attenuated in Dusp6-/- mice 39 3.8 Deficiency of DUSP6 protects mice from acute lung injuries during experimental sepsis 41 3.9 Pulmonary endothelial DUSP6 is essential for LPS-induced neutrophil recruitment in mice 42 3.10 Exploring DUSP6-mediated phosphorylation network in TNF-alpha-activated HUVECs by MS analysis 44 CHAPTER 4: DISCUSSION 48 CHAPTER 5: FUTURE PERPECTIVES 55 CHAPTER 6: FIGURES 59 CHAPTER 7: REFERENCES 96 APPENDIX 104 List of identified phosphoproteins altered in DUSP6-ablated HUVECs | |
| dc.language.iso | en | |
| dc.subject | 肺部損傷 | zh_TW |
| dc.subject | 腫瘤壞死因子alpha | zh_TW |
| dc.subject | 內皮細胞發炎反應 | zh_TW |
| dc.subject | 雙專一性磷酸水解? | zh_TW |
| dc.subject | 細胞表面黏著分子 | zh_TW |
| dc.subject | 白血球 | zh_TW |
| dc.subject | 敗血症 | zh_TW |
| dc.subject | intercellular adhesion molecule-1 (ICAM-1) | en |
| dc.subject | lung injury | en |
| dc.subject | sepsis | en |
| dc.subject | tumor necrosis factor-alpha (TNF-alpha) | en |
| dc.subject | endothelial inflammation | en |
| dc.subject | dual specificity phosphatases 6 (DUSP6) | en |
| dc.subject | neutrophil | en |
| dc.title | 雙專一性磷酸水解酶對於血管內皮細胞發炎反應的調控 | zh_TW |
| dc.title | The Role of Inducible Dual-Specificity Phosphatases in Vascular Endothelial Inflammation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王寧,裘正健,張震東,邱繼輝 | |
| dc.subject.keyword | 腫瘤壞死因子alpha,內皮細胞發炎反應,雙專一性磷酸水解?,細胞表面黏著分子,白血球,敗血症,肺部損傷, | zh_TW |
| dc.subject.keyword | tumor necrosis factor-alpha (TNF-alpha),endothelial inflammation,dual specificity phosphatases 6 (DUSP6),intercellular adhesion molecule-1 (ICAM-1),neutrophil,sepsis,lung injury, | en |
| dc.relation.page | 108 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2015-08-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-104-1.pdf | 7.96 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。