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系統識別號 U0026-0812200910205637
論文名稱(中文) 白血球NADPH氧化酵素在缺血性腦中風致病機轉之角色
論文名稱(英文) Roles of the leukocyte NADPH oxidase in the pathogenesis of ischemic stroke
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 90
學期 2
出版年 91
研究生(中文) 陳彥君
研究生(英文) Yen-Chun Chen
學號 t1689108
學位類別 碩士
語文別 英文
論文頁數 71頁
口試委員 口試委員-張明熙
指導教授-謝奇璋
指導教授-林秀娟
口試委員-江美治
中文關鍵字 缺血性腦中風  動脈粥狀硬化  介白素-22  C反應蛋白  基因多型性  氧自由基  NADPH氧化酵素 
英文關鍵字 Reactive oxygen species (ROS)  Interleukin-22  C-reactive protein (CRP)  Polymorphism  Atherosclerosis  Ischemic stroke  NADPH oxidase 
學科別分類
中文摘要 動脈粥狀硬化(atherosclerosis)是引起缺血性腦中風(ischemic stroke)重要的危險因子。當動脈發生硬化時,在病變處有明顯發炎反應(inflammation)的進行,因此動脈粥狀硬化被認定為一種慢性的發炎反應。氧自由基(reactive oxygen species)在動脈粥狀硬化的發炎反應裡扮演著關鍵性的角色。雖然人體內有許多產生氧自由基的機制,NADPH氧化酵素(NADPH oxidase)仍被認定是最主要的來源。NADPH氧化酵素最早被發現表現在嗜中性白血球上,由多種蛋白質次元(gp91phox, p22phox, p40phox, p47phox, p67phox,以及Rac2)共同組合而成的。近年有研究指出,p22phox基因多型性與血管疾病的發生有關。此外,Interleukin-22(IL-22)是屬於IL-10家族中的一員。相關研究指出,IL-22能刺激白血球產生氧自由基。因此本篇研究想要探討NADPH氧化酵素與缺血性腦中風之間的關聯性。我們收集腦血性腦中風患者以及性別年齡相仿的健康對照者之血液檢體。利用微量化學冷光測量儀(chemiluminescence analyzer)偵測白血球在PMA刺激與否狀態下,產生氧自由基的多寡,並加以比較病人與對照組氧自由基產生量之差異。聚合酵素連鎖反應(PCR)以及限制酵素截切(restriction enzyme digestion)用來判斷病人與對照組在p22phox基因多型性的比例。利用酵素結合免疫吸附法(ELISA),測量病人與對照組血清中一些發炎指標性蛋白質的濃度。我們發現缺血性腦中風的病人較健康人產生更多量的氧自由基。一些複診病人在脫離急性發炎期後仍然持續產生高量的氧自由基。基因方面的分析指出兩種p22phox基因多型性的比例,在病人與對照組之間沒有顯著的差異。相較健康對照組,CRP,MCP-1,和P-selectin這些發炎指標性蛋白質在病人體內有增高的趨勢。IL-22能刺激白血球,使其能產生更大量的氧自由基。這些結果顯示由NADPH氧化酵素產生的氧自由基與缺血性腦中風之間有關。高量氧自由基的表現,可能會經由引發動脈硬化的發炎反應,進一步導致中風的產生。關於NADPH氧化酵素在體內活化的機轉,例如IL-22,其中作用的方式和在疾病中的角色需要更進一步的研究加以探討。
英文摘要 Inflammation has been known to be a key factor in the development of atherosclerosis that leads to ischemic stroke. Reactive oxygen species (ROS) play various roles in the inflammatory process of atherosclerosis. Even though different potential sources of ROS have been found, NADPH oxidase is the most likely source of ROS in tissues. NADPH oxidase, originally characterized in neutrophils, is a multi-component enzyme complex, composed of gp91phox, p22phox, p40phox, p47phox, p67phox and Rac2 proteins. Genetic polymorphisms in p22phox gene were reported to be associated with atherosclerotic vascular diseases. Interleukin-22 (IL-22) is a member of IL-10 family and its functions remain unclear. IL-22 has been found to regulate ROS generation through unknown mechanisms. In this investigation, we sought to investigate whether NADPH oxidase is associated with the pathogenesis of ischemic stroke. Patients with ischemic stroke and control subjects were recruited. Basal and PMA-stimulated superoxide production was measured from neutrophils with a lucigenin-enhanced chemiluminescence method. Polymorphisms in the p22phox gene in patients and control subjects were characterized with PCR and restriction enzyme digestion. Inflammation markers were measured with ELISA. We found that superoxide production from patients was higher than that from control subjects. Amounts of superoxide production from patients in acute and convalescent stages were not significantly different. Proportion of polymorphisms in p22phox between patients and control subjects were not different. Levels of inflammatory markers, including C-reactive protein (CRP), human monocyte chemotactic protein-1 (MCP-1), and P-selectin, were elevated in patients. IL-22 was potent in priming leukocytes to generate more ROS. These results suggested that ROS from NADPH oxidase may participate in the pathogenesis of ischemic stroke through a proinflammatory mechanism. Further studies are needed to investigate the function and regulation of NADPH oxidases and their role in ROS generation in human diseases.
論文目次 English abstract………………………………………………………I
Chinese abstract………………………………………………………III
Table of Contents…………………………………………………… V
List of Tables………………………………………………………… VIII
List of Figures……………………………………………………………IX
List of Appendices……………………………………………………… XI
1. Introduction…………………………………………………………………… 1
1.1 Stroke………………………………………………………………………1
1.2 Atherosclerosis……………………………………………………………………… 1
1.3 Reactive oxygen species (ROS)…………………………………………………… 3
1.4 NADPH oxidase………………………………………………………… 4
1.4.1 Structure and function……………………………………………………………4
1.4.2 Polymorphisms of p22phox…………………………………………………………6
1.5 Cytokines in the pathogenesis of atherosclerosis……………………………7
1.6 Interleukin-22 (IL-22)………………………………………………………………8
1.7 Aims of this study……………………………………………………………………9
2. Material and methods…………………………………………………………………10
2.1 Samples……………………………………………………………………………10
2.2 Leukocyte counts…………………………………………………………………… 10
2.3 Peripheral blood smear preparation…………………………………………… 10
2.4 Isolation of granulocytes and peripheral blood mononuclear cells…… 11
2.5 Superoxide production test by chemiluminescence test…………………… 12
2.6 DNA isolation…………………………………………………………………13
2.7 Polymerase chain reaction…………………………………………………………14
2.7.1 Exon 4 of p22phox…………………………………………………………………15
2.7.2 Exon 6 of p22phox…………………………………………………………………15
2.8 Restriction enzyme digestion…………………………………………………… 16
2.8.1 Exon 4 of p22phox…………………………………………………………………16
2.8.2 Exon 6 of p22phox…………………………………………………………………16
2.9 PCR product purification………………………………………………………… 17
2.10 DNA sequencing…………………………………………………………………17
2.11 Measurement of inflammation markers………………………………………… 18
2.11.1 DueSet ELISA Development System…………………………………………… 18
2.11.1.1 Plate preparation…………………………………………………………… 18
2.11.1.2 Assay procedure……………………………………………………………… 19
2.11.2 Parameter ELISA system…………………………………………………………20
2.11.2.1 Reagent preparation………………………………………………………… 20
2.11.2.2 Assay procedure……………………………………………………………… 21
2.12 Priming effects of IL-22…………………………………………………………22
2.13 Statistical analysis…………………………………………………………… 23
3. Results…………………………………………………………………………… 24
3.1 Clinical characteristics of ischemic stroke and control subjects…… 24
3.2 Chemiluminescence measurements reflex ROS production of
leukocytes…………………………………………………………………………… 24
3.3 Superoxide production from granulocytes was much higher than that
from peripheral blood mononuclear cells………………………………………25
3.4 Superoxide production was higher in patients with ischemic stroke……26
3.5 Restriction enzyme digestion of PCR products of exon 4 and exon 6 of
p22phox from patients and controls revealed gene polymorphisms……… 26
3.6 DNA sequencing confirmed the results from restriction enzyme digestion………………………………………………………………………………27
3.7 Proportions of two single nucleotide polymorphisms of p22phox were
not different between patients and controls…………………………………28
3.8 No correlation between A640G polymorphism and superoxide
production was observed……………………………………………………………28
3.9 Several inflammation markers were increased in patients…………………28
3.9.1 CRP………………………………………………………………………………29
3.9.2 MCP-1……………………………………………………29
3.9.3 Soluble P-selectin……………………………………………………………… 30
3.9.4 Soluble E-selectin……………………………………………………………… 30
3.9.5 Soluble ICAM-1……………………………………………………………… 30
3.9.6 Soluble VCAM-1……………………………………………………………… 31
3.10 Relationship between CRP levels and superoxide production in patients
with ischemic stroke………………………………………………………………31
3.11 IL-22 is potent in priming leukocytes to generate more superoxide……32
4. Discussion………………………………………………………………………33
5. References………………………………………………………………………42
Tables……………………………………………………………………… 48
Figures………………………………………………………………………51
Appendices………………………………………………………………………… 69
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