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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 呂紹俊(Shao-Chun Lu) | |
dc.contributor.author | Tzu-Ching Yang | en |
dc.contributor.author | 楊子慶 | zh_TW |
dc.date.accessioned | 2021-06-17T02:11:52Z | - |
dc.date.available | 2018-02-22 | |
dc.date.copyright | 2018-02-22 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2018-01-04 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68054 | - |
dc.description.abstract | L5-low-density lipoprotein (L5-LDL) 是帶有陰電性的低密度脂蛋白,存在於高膽固醇血症、第二型糖尿病與急性心肌梗塞患者的血液中。臨床研究發現L5-LDL在ST時段上升心肌梗塞(STEMI)病人血液中有顯著增加的情形,同時血液中的介白素-1β (Interleukin-1β, IL-1β)、顆粒球群落刺激因子(granulocyte colony-stimulating factor, G-CSF)及顆粒球巨噬細胞群落刺激因子(granulocyte macrophage colony-stimulating factor, GM-CSF)也有明顯的上升,而這些細胞激素的產生和急性心肌梗塞的嚴重程度有所關連。發炎被認為是造成心血管疾病最主要的因素之一,但什麼因子引起這些發炎反應及細胞激素的產生仍不清楚。因此本篇論文探討由心肌梗塞病人中分離出的陰電性低密度脂蛋白L5-LDL是否會引起IL-1β、G-CSF及GM-CSF的產生及其相關機制。
本篇論文包含兩部分,第一部分探討L5-LDL造成促發炎性細胞激素IL-1β的產生及機制。在人類周邊血球單核球細胞(human peripheral blood mononuclear cells, PBMC)及人類單核球細胞株(THP-1)所分化的巨噬細胞中,有別於不帶電的L1-LDL或銅離子氧化的Cu-oxidized LDL (Cu-oxLDL),從ST時段上升心肌梗塞病人中分離的L5-LDL顯著增加了IL-1b的分泌及半胱天冬酶 (caspase-1)的活化。以short hairpin (shRNA)減少NOD-like receptor pyrin domain containing 3 (NLRP3)發炎體(inflammsome)主體蛋白NLRP3和apoptosis-associated speck-like protein containing a CARD (ASC)的表現,可以顯著減少L5-LDL所產生的IL-1β,顯示L5-LDL促進IL-1β的分泌是經由活化NLRP3發炎體。而在shRNA減少類凝集素氧化低密度脂蛋白受器-1(lectin-like oxidized low-density lipoprotein receptor-1, LOX-1)表現的THP-1細胞,L5-LDL所活化的NF-κB及caspase-1及IL-1b的都明顯的低於轉染冷光酶(luciferase) shRNA的對照組THP-1細胞。此外於PBMC分化的巨噬細胞中預先處理LOX-1中和抗體TS92或 NF-kB 抑制劑Bay11-7082,可顯著降低IL-1β的表現。這些結果顯示L5-LDL在巨噬細胞透過LOX-1及發炎體訊息途徑促進IL-1β產生。 第二部分則探討L5-LDL在巨噬細胞促使G-CSF及GM-CSF產生的機制,結果顯示L5-LDL在PBMC及THP-1所分化的巨噬細胞中促進G-CSF及GM-CSF的產生,而L1-LDL和Cu-oxLDL則無明顯的作用。進一步將L1-LDL及L5-LDL以銅離子氧化後會影響其產生G-CSF及GM-CSF的能力,顯示氧化低密度脂蛋白引起巨噬細胞發炎的作用與其氧化程度有關。利用shRNA抑制LOX-1,可以發現L5-LDL所誘導的G-CSF及GM-CSF大幅減少,顯示L5-LDL透過LOX-1 促進G-CSF及GM-CSF的產生。若預先處理NF-κB抑制劑Bay-117082及extracellular signal-regulated kinase 1/2(ERK1/2)磷酸化抑制劑U0126,可有效抑制L5-LDL所誘導的G-CSF和GM-CSF。此外在利用shRNA 抑制ERK1和ERK2的THP-1細胞,僅有抑制ERK2的細胞會減少L5-LDL所誘導產生的G-CSF和GM-CSF。結果顯示L5-LDL透過LOX-1、ERK2 和NF-κB途徑促進G-CSF及GM-CSF的產生。 在本篇論文中我們也闡述了STEMI L5-LDL在巨噬細胞促進IL-1β、G-CSF及GM-CSF產生的可能機制,這些因子都與發炎有關,因此L5-LDL可能是急性心肌梗塞時引起發炎的重要因子。 | zh_TW |
dc.description.abstract | L5-LDL, the most electronegative LDL, is associated with major cardiovascular risks, and which is significantly higher in the plasma of patients with ST-segment elevation myocardial infarction (STEMI). The inflammatory response of atherosclerotic vascular diseases has prompted us to investigate whether L5-LDL induces the production of inflammatory cytokines, especially vascular ischemia-related interleukin (IL)-1β in the pathogenesis of STEMI. Besides, circulating levels of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF are also associated with the severity of acute myocardial infarction (AMI). However, what causes the increases in IL-1β, G-CSF and GM-CSF is unclear. In this study, we investigated whether production of IL-1β, G-CSF and GM-CSF in human macrophages is induced by L5-LDL, an inflammatory activator in AMI.
This study consists of two parts. The first part demonstrated the link of L5-LDL and the production of inflammatory cytokine IL-1β in STEMI. L5-LDL significantly increased the protein levels of both IL-1β and cleaved caspase-1 in THP-1 derived human macrophages, indicating the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes by L5-LDL. Knockdown of NLRP3 and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) resulted in decreased L5-LDL-induced IL-1β. Furthermore, knockdown of the lectin-type oxidized LDL receptor-1 (LOX-1) in THP-1 cells attenuated L5-LDL-induced activation of nuclear factor (NF-κB) and caspase-1, leading to subsequent inhibition of IL-1b production in macrophages. Furthermore, blockade LOX-1 with neutralizing antibody also inhibited L5-LDL-induced IL-1β in human peripheral blood mononuclear cells derived macrophages (PBMC). In conclusion, L5-LDL induces IL-1β production in macrophages by activation of NF-κB and caspase-1 through the LOX-1 dependent pathway. In the second part, we investigated whether production of G-CSF and GM-CSF in human macrophages is induced by L5-LDL. Treatment with L5-LDL, but not L1-LDL or copper oxidized LDL, induced production of both G-CSF and GM-CSF in THP-1 macrophages. In vitro oxidation of L1-LDL and L5-LDL altered their ability to induce G-CSF and GM-CSF, suggesting that the degree of oxidation is critical for the effects. Knockdown experiments showed that the effects were significantly inhibited in LOX-1- but not in CD36-knockdown cells. Knockdown and antibody neutralization experiments suggested that the effects were mediated by LOX-1. In addition, NF-κB and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibition resulted in marked reductions of L5-LDL-induced G-CSF and GM-CSF production. Moreover, knockdown of ERK2, but not ERK1, hindered L5-LDL-induced G-CSF and GM-CSF production. The results indicate that L5-LDL induced G-CSF and GM-CSF production in human macrophages through LOX-1, ERK2, and NF-κB dependent pathways. We therefore propose a novel mechanism underlying the inflammatory response in AMI. Taken together, this study identifies the mechanisms that L5-LDL induced production of IL-1β, G-CSF and GM-CSF in macrophages. All these factors are related to inflammation and thus suggest L5-LDL is a critical factor that provoke inflammation in STEMI. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:11:52Z (GMT). No. of bitstreams: 1 ntu-106-D02442001-1.pdf: 3865126 bytes, checksum: 243257449b5af09fd70119398d7bf439 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員會審定書 i
摘 要 ii Abstract v List of abbreviations viii List of Figures xiii List of Tables xiv Chapter I. Introduction 1 Acute myocardial infarction 1 Atherosclerosis and oxidized LDL 1 Characteristics of electronegative LDL 3 The role of L5-LDL in cardiovascular disease 4 Association between STEMI and IL-1β 5 NLRP3 inflammasome and oxLDL 8 The role LOX-1 in AMI 10 The roles of G-CSF and GM-CSF 11 Elevation of G-CSF and GM-CSF in AMI 12 Aims of the study 13 Chapter II. Materials and Methods 15 2.1 Materials 15 2.2 Methods 16 2.2.1 Study subjects 16 2.2.2 Cell culture and lipoprotein treatment 17 2.2.3 LDL preparations 19 2.2.4 Quantification of IL-1β in patient plasma and culture media 20 2.2.5 Quantification of G-CSF and GM-CSF in culture medium 20 2.2.6 Western blot analysis 20 2.2.7 RNA isolation and reverse-transcription qualitative polymerase chain reaction (RT-qPCR) 22 2.2.8 Knockdown of CD36, LOX-1, NLRP3, ASC, ERK1 and ERK2 by RNA interference 23 2.2.9 Analysis of NF-κB activity 24 2.2.9 Immunohistochemical analyses 25 2.2.10 Cell viability MTT Assay 26 2.2.11 Statistical analysis 26 Chapter III. Results 27 Part I. L5-LDL from STEMI patients induces IL-1β production via LOX-1 and NLRP3 inflammasome activation in macrophages 27 3.1.1 L5-LDL stimulates L5-LDL from STEMI patients induces IL-1β production via LOX-1 and NLRP3 inflammasome activation in macrophages inflammatory cytokines in THP-1 macrophages 27 3.1.2 Plasma L5-LDL and IL-1β levels are significantly elevated in STEMI patients 28 3.1.3 L5-LDL induces IL-1β production in THP-1 macrophages 28 3.1.4 L5-LDL-induced IL-1β production is caspase-1 dependent 29 3.1.5 NLRP3 inflammasome is involved in L5-LDL-induced IL-1β production 30 3.1.6 Copper oxidation of L5-LDL attenuates L5-LDL-induced IL-1β production 31 3.1.7 LOX-1 mediates L5-LDL-induced IL-1β production in macrophages 32 3.1.8 NF-κB is involved in L5-LDL-LOX-1 signaling for IL-1β production 34 3.1.9 L5-LDL induces IL-1β activation through LOX-1 and NFκB in primary human macrophages 36 3.1.10 Summary 37 Part II. Electronegative L5-LDL induces production of G-CSF and GM-CSF in human macrophages via LOX-1 and involving NF-κB and ERK2 activation 38 3.2.1 L5-LDL induces G-CSF and GM-CSF production in THP-1 macrophages 38 3.2.2 The oxidation statuses of L1-LDL and L5-LDL affects their ability to induce G-CSF and GM-CSF production 39 3.2.3 L5-LDL induces G-CSF and GM-CSF production through a LOX-1-dependent pathway 40 3.2.4 Inhibition of ERK1/2 and NF-κB activation prevents L5-LDL-induced production of G-CSF and GM-CSF 41 3.2.5 ERK2 mediates L5-LDL-induced production of G-CSF and GM-CSF 43 3.2.6 L5-LDL induces G-CSF and GM-CSF production in primary human macrophages 43 3.2.7 Summary 44 Chapter IV. Discussion 45 Part I. L5-LDL from STEMI patients induces IL-1β production via LOX-1 and NLRP3 inflammasome activation in macrophages 45 Part II. Electronegative L5-LDL induces production of G-CSF and GM-CSF in human macrophages via LOX-1 and involving NF-κB and ERK2 activation 49 Chapter V. Overall Conclusion and Perspective 54 Chapter VI. Figures 57 Chapter VII. Tables 94 References 96 List of publications 119 | |
dc.language.iso | en | |
dc.title | 急性心肌梗塞患者血中陰電性低密度脂蛋白引發巨噬細胞之發炎反應 | zh_TW |
dc.title | Electronegative low-density lipoprotein from acute myocardial infarction patients induces inflammation in macrophages | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 張博淵,張淑芬,黃青真,李啟明,李安生 | |
dc.subject.keyword | 介白素-1β,類凝集素氧化低密度脂蛋白受器-1,粒細胞集落刺激因子,粒細胞巨噬細胞集落刺激因子,陰電性低密度脂蛋白,巨噬細胞,急性心肌梗塞, | zh_TW |
dc.subject.keyword | IL-1β,electronegative LDL,LOX-1,STEMI,G-CSF,GM-CSF,macrophage,acute myocardial infarction, | en |
dc.relation.page | 120 | |
dc.identifier.doi | 10.6342/NTU201704523 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-01-05 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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