進階搜尋

 
查詢範圍:「   」
顯示範圍:第筆 論文書目資料
顯示格式:全部欄位
共 19 筆
------------------------------------------------------------------------ 第 1 筆 ---------------------------------------------------------------------
系統識別號 U0007-0107201021062700
論文名稱(中文) 醫學人文劇『幸福的記憶』社會實踐之研究
論文名稱(英文) Medical humanities drama, The Memory of Happiness – the study of social practice
校院名稱 臺北醫學大學
系所名稱(中) 醫學人文研究所
系所名稱(英) Graduate Institute of Humanities in Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 陳永旭
學號 M116097005
學位類別 碩士
語文別 中文
口試日期 2010-06-25
論文頁數 179頁
口試委員 指導教授-陳永興
指導教授-蔡篤堅
委員-洪傳岳
委員-陳克華
委員-曾賢熙
關鍵字(中) 醫學人文劇
失智症
媒體論述
生命敘事
關鍵字(英) medical humanities play
dementia
media discourse
life narrative
學科別分類
中文摘要 研究者從媒體對失智症觀點處理如何影響社會主流的刻板印象,到障礙社會學、老人社會學等對建構大眾的失智症觀點的批判及如何應用劇場的生命敘事理論和行動研究的方法扭轉現今社會不恰當的對失智症議題的關注方式,以解說、重現失智症患者及其照顧者的主體性,使得失智症的議題能夠不被媒體、社會福利機構、大眾的既定印象局限,用多元化的角度詮釋失智症議題,真實地呈現失智症患者所面臨的生命困境與實際上對有限的餘生仍有無限的可能,還有照顧者真實所需要的社會支持和資源等等問題;透過劇場的展演這一過程,使傳統的失智症標籤和印象徹底地得到解放,因為研究者希望製作及導演以『病人』的病痛及以生命探討為對象的作品,讓台灣的醫學教育是以培養具備悲天憫人胸懷的良醫為目標。
醫師治病不是為了自己,也不是為了利慾,而是為了解除人的痛苦,恢復人的健康,增進人的福祉為首要任務。研究者希望透過醫病關係戲劇的創作理念傳達與分享,為台灣醫學人文教育紮根,讓第一線的醫療工作者,及未來即將從事醫護工作的學生們,透過「醫學人文劇」的戲劇精神,能更了解醫病關係,也讓醫護學生藉由醫療專業角色的扮演,傳達醫療工作者面對疾病的態度及生命觀點,由真實案例去反思生命的意義,並能更懂得及時去愛惜親人和活在當下!
期望本研究對於阿茲海默氏症患者與主要照顧者面對疾病過程的心路歷程有一個新的理解,以提供失智家屬、民眾和醫療人員一個新的觀點,讓失智長者都能獲得尊嚴的對待與關懷。
關鍵字:醫學人文劇、失智症、媒體論述、生命敘事

英文摘要 Abstract
Researchers from the media point of view on dealing with dementia, how stereotypes affect social mainstream, to the obstacles to sociology, sociology elderly dementia views on the construction of public criticism and the application of narrative theater of life theories and research methods reverse the current mainstream is not the appropriate way on dementia issues concerned to explain, to reproduce the dementia patients and their caregivers subjectivity, so the question of dementia can not be the media, social welfare agencies, public the impression given the limitations of the angle of dementia with diversity issues, truly showing the life of dementia patients face difficulties with the rest of his life still in effect limited the possibility of unlimited, there are real needs to take care of social support and resource problems; through theater performances in this process, the traditional label of dementia and impressions thoroughly liberated, as the researchers hope to make, and the director are illness and the pain of life to work for the object, Taiwan's medical education is to cultivate the good doctor with compassion in mind the goal.
Physicians not to treat themselves, nor for acquisitiveness, but to relieve human suffering and restore people's health, enhance well-being top priority. The researchers hope that the doctor-patient relationship through the creation of drama to convey ideas and sharing of medical humanities education rooted in Taiwan, so that the first-line medical workers, and the future will be working in the care of students, through the "medical humanities drama" dramatic spirit can better understand the relationship between doctors and patients, but also for medical students to the role played by the medical profession, to convey the attitude of health workers in the face of illness and life view, by the true story to reflect on the meaning of life and more time to cherish loved ones and know how to Live in the moment!
Finally, this study gives a deep and new insight of the track of lives of Alzheimer's disease sufferers and their nurses. It provides the family of patients with dementia, the public and medical professionals with a new point of view so that patients with dementia can be treated carefully with affection and dignity.

Keywords: medical humanities play, dementia, media discourse, life narrative
論文目次 目錄
第一章 緒論…………………………………………………………1
第一節 研究動機………………………………………………………………1
第二節 研究背景………………………………………………………………4
一、 失智症的定義與簡介……………………………………………5
二、 台灣失智症人口趨勢……………………………………………6
三、 失智症患者的需求與社會福利供給現況………………………7
四、 失智症照顧者無法取得社會福利資源…………………………8
五、 分析:照顧體系對失智症患者的選擇性遺忘…………………9
第三節 研究目的...............................................12

第二章 文獻探討…………………………………………13
第一節 社會大眾對失智症的標籤印象.............................14
一、 傳播媒體對弱勢族群的再現政治.......................15
二、 老人社會學的標籤印象...............................17
三、 障礙社會學的詮釋方法...............................18
第二節 失智症的醫療盲點.......................................20
一、 傅柯談疾病的規訓與汙名.............................20
二、 疾病敘事...........................................25
第三節 生命敘事的可能性.......................................32
一、 透過生命敘事重構失智症議題.........................32
二、 以劇場作為一種新的療癒形式.........................33
第三章 研究方法…………………………………………………37
第一節 研究架構...............................................37
第二節 研究方法與研究內容.....................................38
一、 論述分析...........................................38
二、 田野工作...........................................39
三、 行動研究...........................................40
第三節 研究流程與實施步驟.....................................43
第四章 媒體分析………………………………………46
第一節 媒體的批判論述分析研究---揭發絃外之音………………………46
第二節 分析一:媒體中的失智症的再現印象………………………………46
分析二:媒體中的「孝道文化」變遷與影響………………………66

第五章 研究結果……………………………………………………74
第一節 障礙研究新視野.........................................74
第二節 媒介的再詮釋---生命敘事理論.............................79
第六章 行動研究的實踐與討論…………………………………81
第一節 醫學人文劇的興起背景...................................81
第二節 劇場實踐回顧---明日的記憶..............................82
一、 劇本緣起...........................................82
二、 劇本主軸:陪伴與珍貴的記憶.........................84
三、 劇本分析...........................................84
四、 詮釋主體性—從演員與病患的訪談重構失智症意象.......88
第三節 實踐成果與討論.........................................92
一、 從電影《明日的記憶》到舞台劇《幸福的記憶》.........92
二、 演出迴響─各場次現場概況...........................98

第七章 結論與建議………………………………………………105
第一節 研究結論..............................................109
第二節 研究限制..............................................109
第三節 研究建議……………………………………………………………109
參考文獻………………………………………………………………………112

附錄

附錄一演出紀實…………………………………………………………119

附錄二觀眾迴響與反應………………………………………………………126

附錄三觀眾意見調查表………………………………………………………135

附錄四媒體報導……………………………………………………………………136

附錄五演出劇本……………………………………………………………………137



參考文獻 參考文獻
一、中文書目
片桐新自、永井良和、山本雄二,《基礎社會學》,蘇碩斌、鄭陸霖譯,台北:群學出版社,2008。
王增勇:《老年與反抗:美國老人運動背後的主體》,《傅柯與社會工作》,233頁,台北,心理出版社,2006年8月二版。
Catherine E. Foote and Arthur W. Frank著:《傅柯與治療:悲傷的規訓》,《傅柯與社會工作》,王增勇、簡憶鈴、譯,台北,心理出版社,2006年8月二版。
王墨林:《都市劇場與身體》,台北,稻鄉,1992年。
石世明:《伴你最後一程:臨終關懷的愛與慈悲》,台北,天下遠見,2001年。
李宇宙:《疾病的敘事與書寫》,《人文、醫學與疾病敘事》,台北,記憶工程、唐山,2007年。
克莉絲汀•伯頓:《親愛的,你記得我是誰嗎?:一位阿茲海默氏症患者的生命之歌》,李素卿譯,台北,傳神,2004年。
邱天助,《年齡歧視與年齡解放:老人不是社會問題》世新大學社會心理學系,2002。
杏林子:《杏林小記》,台北,九歌,1979年3月。
林偉瑜:《當代台灣社區劇場》,台北,揚智文化,2000年。
夏林清等譯:《行動研究方法導論:教師動手作研究》,台北,遠流,2000年。
夏曉鵑,《流離尋岸:資本主義國際化下的「外籍新娘」現象》,2002。
孫華瑛:《社區劇場與女性賦權》,《劇場事》,台南,台南人劇團,2009年7月。
孫家棟,《失智症之行為精神症狀及照顧者負擔研究》,合記醫學,2000。
馬修•奈松斯等著:《阿茲海默診療室》(There's Still A Person in There),易之新譯,2001年2月。
張世平:《行動研究法》,《教育研究法》黃光雄、簡茂發編,台北,師大書苑,1991年。
郭佩宜、王宏仁主編:《田野的技藝:自我、研究與知識建構》,台北巨流圖書公司,2007年。
陳榮基,《失智症照護指南》,合記醫學,1990。
麥可•懷特(Michael White)、大衛•艾普斯頓(David Epston):《故事、知識、權力─敘事治療的力量》,廖世德譯,台北,心靈工坊,2001年4月。
梅洛•龐蒂:《知覺現象學》,姜志輝譯,北京市,商務印書館,2001年。
黃政傑:《課程改革》,台北,漢文,1999年。
凱博文(Arthur Kleiman):《談病說痛─人類的受苦經驗與痊癒之道》,台北,桂冠,1994年。
雄恩•麥克尼夫(Shaun McNiff):《藝術治療》,許邏灣譯,台北,新路,1999年6月。
歐文•亞隆:《存在心理治療─自由、孤獨、無意義》,易之新譯,台北,張老師文化,2003年。
歐文•亞隆:《當尼采哭泣時》,侯維之譯,台北市:張老師文化,2000年。
賴淑雅:《台灣社區劇場的發展脈絡與核心價值》,《劇場事》,台南,台南人劇團,2009年7月。
賴淑雅:《在劇場中看見自己與社會〉,《劇場事》,台南,台南人劇團,2009年7月。
賴淑雅主編:《區區一齣戲:社區劇場理念與實務手冊》,台北,文化建設委員會,2006年。
Augusto Boal:《被壓迫者劇場》(Theatre of the Oppressed),賴淑雅譯,台北,揚智,2000年。
蘇珊•桑塔格:《疾病的隱喻》,刁筱華譯,台北,大田出版社,2000年。(原著出版年:1997)
赫伯特•馬庫色(Herbert Marcuse):《反革命與反叛》,高志仁譯,台北,立緒文化,2000年8月。
鍾喬:〈蝸牛,不見得比鳥慢─當民眾劇場遇上成人教育〉,《劇場事》,台南,台南人劇團,2009年7月。
鍾明德:《現代戲劇講座:從寫實主義到後現代主義》,台北,書林,1995年。
謝國雄主編:《以身為度、如是我做:田野工作的教與學》,台北,群學,2007年。
圖姆斯(S.K Toombs):《病患的意義——醫生和病人不同觀點的現象學探討》,青島,青島出版社,2000年6月。

二、期刊文章

王浩威〈尋找自己觀眾的劇場(上)(下)—評《亞洲的吶喊》一書兼談台灣小劇場的策略問題〉,《表演藝術》,1994年。
王釧如:〈與羈絆的靈魂共舞─一位阿茲海默氏症患者之主要照顧者的生命經驗敘事〉,南華大學生死學系碩士論文,2008年。
史曉寧, 黃愛娟(1996), 〈家庭重病老年患者之主要照顧者壓力源和其家庭功能因素之探討〉, P.138-147.
成令方:〈醫用關係的知識與權力〉,《台灣社會學》,NO.3,2002年。
呂寶靜,〈老人照顧:老人、家庭、正式服務〉,台大社工學刊,2001。
邱玉蟬〈醫病形象的媒體建構》長庚大學醫務管理學研究所,2007。
邱啟潤, 許淑敏, 吳淑如(2003), 〈居家照護病患之主要照顧者綜合性需求調查〉, 《醫護科技學刊》, P.12-25.
李薇,〈失智症老人社會心理適應的護理人類學研究〉第8頁,慈濟大學人類學研究所碩士論文,2007。
李欣倫:〈戰後台灣疾病書寫研究〉,國立中央大學中國文學研究所碩士論文,1993年。
林國明,〈朝向醫療社會學創造性的對話與整合〉,台灣社會學第三期,台北。
林健群,〈失智老人社會心理適應的護理人類學研究》,慈濟大學人類學研究所碩士論文,1998。
林文源:〈To Be or Not To Be─長期洗腎病患的生活、身體經驗》,新竹,清華大學社會人類學研究所碩士論文,1998年。
倪炎元,〈再現的政治:解讀媒介對他者負面建構的策略〉,《新聞學研究》,1999。翁秀琪〈批判語言學、在地權力觀和新聞文本分析〉,新聞學研究第57期頁93,1988。
徐明珠:〈行動研究在教育改革中的問題和價值〉,國家政策論壇,2004年。

張笠雲:〈「逛醫師」的邏輯:求醫歷程的分析》,台北,台灣社會學刊第二十一期,1998年。
張恆豪、蘇峰山,〈戰後台灣國小教科書中的障礙者意象分析〉,〈台灣社會學刊》,42:143-188,2009。
張恆豪,〈特殊教育與障礙社會學一個理論的反省〉,〈教育與社會研究〉,71-93,2007。
陳明珠,〈媒體再現與認同政治》,中華傳播學會論文,2002。
陳昱名,〈社會福利資源與需求的落差:以老年失智症病患家庭照顧者之照顧負荷,需求與困難為例〉,台北醫學大學醫學研究所,2003。
許禮安:〈病情世界的多重現象分析〉,國立東華大學族群關係與文化研究所碩士論文,2005年。
葉光輝,〈華人孝道的心理與行為〉,華人本土心理學刊305-308頁,2005。
葉光輝,〈孝道困境及其消解模式》中央研究院民族學研究所集刊 79:87-118,1995。
關晨引:〈劇場與社會改造:戲劇工作方引導者的實作〉,世新大學社會發展研究所碩士論文,2009年7月。
蕭慧文〈吸引人的身體-從梅洛龐蒂和葛羅托斯基談表演身體的主體性》,臺北藝術大學戲劇研究所碩士論文,2002年。
蕭阿勤,〈回歸現實:臺灣1970年代的戰後世代與文化政治變遷〉,台北:中央研究院社會學研究所,2008。
劉徖琦,〈社會弱勢者的媒體形象研究:以外籍家事勞動者與顧煮關係之報導〉頁4-6,中華傳播學會,2008。
蔡友月:〈台灣癌症病患的身體經驗:病痛、死亡與醫療專業權力〉,〈台灣社會學刊〉,第33期,2004年12月。
謝偉姝〈公共電視原住民記者報導觀點之研究》,文化大學新聞研究所碩士論文,1996。

三、英文書目
Atweh, B. , Kemmis, S. , & Weeks, P. (Eds.)(1998) Action research in practice. New York: Routledge.
Bryan S. TURNER (1996) The Blackwell Companion to Social Theory. Oxford, UK:Cambridge, Mass.
European Archives of Psychiatry and Clinical Neuroscience 249 (6): 288–290.
Foucault, Michel. (1975) The birth of the clinic: An archaeology of medical.
Foucault, Michel. (1977) Discipline and punish. London: Allen Lane..
.Foucault, Michel. (1982) The Subject and power. In H. L. Dreyfus & P. Rabinow(Eds.).
Foucault, Michel. (1988) The ethic of care for the self as a practice of freedom. In
Foucault, Michel. (1991) The Foucault effect: Studies in governmentality. Edited by
Forstl H, Kurz A (1999). "Clinical features of Alzheimer's disease".
Kemmis, S. ; MeTaggart, R. (1988), The action research planner (3rd ed.), Victoria: Deakin University Press.
Graham Burchell, Colin Gordon, and Peter Miller. Lodon: Harvester Wheatsheaf.
James Bernauer and David Rasmussen, eds., The final Foucault, pp. 1-20. Cambridge, Mass.: MIT press.
Michael Foucault: Beyond structuralism and hermeneutics. Chicago: Chicago University Press.
Oliver, Michael,The Politics of Disablement. London: The MacMillan Press,1990。
Translated by A. M. Sheridan Smith. New York: Vintage.
.Parsons, Talcott.(1951) The Social System. Glencoe: Free Press.
Scott, Robert,The Making of Blind Men. London. Sage,1969。
Van Dijk 《Structures of News in the Press.》1988。
Willis, Paul(2000) The Ethnographic Imagination. Cambridge: Polity.
Zola, Irving K,.“Toward the necessary universalizing of disability policy," The Milbank Quarterly. 67: 410-428,1989。

四、線上資料
天主教失智老人社會福利基金會http://www.cfad.org.tw/
台灣失智症協會http://www.tada2002.org.tw/
行政院衛生署 委託台灣失智症協會「台灣失智症機構照顧需求之調查長期照護機構失智症患者之盛行率調查」研究報告http://www.tada2002.org.tw/
http://www.alz.org/living_with_alzheimers_chinese_10_signs.asp。
聯合新聞網知識庫http://udndata.com/
【2009-07-08/聯合報/B1版/北市.運動記者袁志豪】
【2009-03-15/聯合報/C1版/北市.教育記者袁志豪】
【2009-05-30/聯合報/B1版/北市.運動/記者姜炫煥】
【2009-03-08/聯合晚報/B6版/健康一起來/記者韋麗文】
【2009-06-30/聯合報/B2版/雲嘉綜合新聞/記者唐秀麗】
【2009-03-27/聯合報/C2版/大台中綜合新聞/記者洪敬浤】
【2009-11-28/聯合報/B2版/北市綜合新聞/記者邱瓊玉】
www.scu.edu.au/schools/gcm/ar/arp/aandr.html
http://blog.chinatimes.com/senior/archive/2007/05/01/162240.html
http://blog.chinatimes.com/senior/archive/2007/05/01/162240.html


------------------------------------------------------------------------ 第 2 筆 ---------------------------------------------------------------------
系統識別號 U0007-0108201011161900
論文名稱(中文) 應用文獻自動化分群系統對於臨床藥學文獻資料檢索服務之探討
論文名稱(英文) An Investigation of Automatic Literature Clustering System for Clinical Pharmacy Literature Searching
校院名稱 臺北醫學大學
系所名稱(中) 醫學資訊研究所
系所名稱(英) Graduate Institute of Biomedical Informatics
學年度 98
學期 2
出版年 99
研究生(中文) 夏景岳
學號 M110093002
學位類別 碩士
語文別 中文
口試日期 2010-07-01
論文頁數 97頁
口試委員 指導教授-蔣以仁
委員-李元綺
委員-劉立
關鍵字(中) 科技接受模式
資訊檢索
群聚分析
自動分群系統
關鍵字(英) Technology acceptance model
Information Retrieval
Clustering Analysis
Automatic Clustering System
學科別分類
中文摘要 隨著網路發達與資訊大量的產出,如何快速有效的在大量資訊中找到問題的解答已經是非常重要的議題。文獻分群技術日益進步的同時,本研究以科技接受模式(Technology acceptance model, TAM)為基礎,透過引介藥師使用自動分群搜尋引擎這項新科技,採用問卷分析的方法探討藥師在使用時的接受程度,進而了解影響藥師使用自動分群搜尋引擎之相關因素。

本研究針對北部地區執業藥師發出四百七十五份問卷,有效回收問卷一百六十三份,結果發現,藥師個人的電腦自我效能以及對於系統認知有用性兩個因素與使用意願有顯著正相關。藥師的年齡、學歷及使用電腦查詢文獻頻率差異與個人電腦自我效能有顯著相關,並直接或間接影響使用的意願。研究結果亦顯示藥師的認知易用性與使用自動化分群系統服務的意願並無顯著正相關。
英文摘要 Since the era of internet arrived, more and more information has been quickly generated, therefore, it’s been a critical issue in how to effectively find the desired content within a great amount of information on the internet. This study is based on Technology acceptance model (TAM) and introducing pharmacists to use automatic clustering search engines. Analyzing the related factors in what affect the pharmacists to use automatic clustering search engines by means of a questionnaire approach.
In this study we investigate pharmacists working in the northern region of Taiwan Island, and the number of valid questionnaires is 163. The study indicates that the Pharmacist’s computer self-efficacy and perceived usefulness have positive and significant influences on behavior intention. Pharmacist's age, education and the use of computer search literature frequency were significantly related to self-efficacy, and affect the use of will. No significant correlation has been found between pharmacist’s perceived ease of use and behavior intention of clustering system services.
論文目次 標題 i
審定書 ii
上網授權書 iii
國科會授權書 iv
誌謝 v
目錄 vi
表目錄 viii
圖目錄 x
中文摘要 xi
英文摘要 xiii
第一章 緒論
1.1 研究背景與動機 1
1.2 研究目的 4
第二章 文獻探討與相關研究
2.1 資訊檢索(Information Retrieval) 7
2.2 特徵選取(Feature Selection) 9
2.2.1 詞性標示(Part of Speech Tagging) 9
2.2.2 專有名詞辨識(Named Entity Recognition, NER) 9
2.2.3 特徵選取(Feature Selection) 12
2.3 關聯法則(Association Rules) 13
2.4 群聚分析(Clustering Analysis) 14
2.4.1 文件相似度 15
2.4.2 分割式群集演算法 16
2.4.3 階層式群集演算法 17
2.5 科技接受模式理論(Technology Acceptance Model,TAM) 17
2.5.1 理性行動理論(TRA) 17
2.5.2 科技接受模型(TAM) 19
2.5.3 電腦自我效能(Computer self-efficacy) 20
2.6 使用自動分群技術網站 21
第三章 研究方法與步驟
3.1 研究架構 28
3.2 研究假設 30
3.3 研究變項與操作定義 31
3.4 問卷設計 31
3.5 研究範圍與對象 35
3.6 資料分析方法 36
第四章 資料分析與結果
4.1 樣本基本資料分析 38
4.1.1 問卷回收概況 38
4.1.2 基本資料之敘述統計分析 39
4.1.3 問卷構面之敘述統計分析 48
4.1.4 開放性問題之敘述統計分析 50
4.2 問卷信度與效度分析 52
4.3 單因子變異數分析. 53
4.4 相關分析. 75
4.5 路徑分析 77
4.6 假設驗證結果 80
第五章 結論與建議
5.1 研究結論 81
5.2 研究限制 86
5.3 後續研究建議 87
參考資料
中文文獻 89
英文文獻 90
電子資料 91
附錄
附錄一 「藥師對文獻自動化分群系統接受程度相關因素探討」之問卷 93
參考文獻 卜小蝶,陳思穎(民 96)。網路自動分群搜尋引擎之使用者評估研究。圖書資訊學研究 2(1),55-80。
王曉璿、楊國賢、盧文偉(民95)。國小學生應用學習管理平台科技接受度探究。Tanet 2006。國立花蓮教育大學,Nov.1-3。
林重志(民95)。實習醫學生在醫學網路資源的資訊尋求及資料庫使用行為之研究。雲林科技大學資訊管理系碩士論文,未出版,雲林縣。
李祐陞(民95)。生醫文獻自動化分群系統與評估。國立台灣大學醫學工程學研究所碩士論文,未出版,台北市。
吳文雄(民 91)。電腦技能學習者過去的績效、目標認同、電腦自我效能及電腦績效因果關係之驗證-社會認知理論與目標設定理論的整合。師大管理學報科學教育類,47(1),39-54。
吳明隆(民 98)。SPSS操作與應用:問卷統計分析實務。台北市:五南圖書公司。
邱皓政(民 89)。量化研究與統計分析---SPSS 中文視窗版資料分析範例解析。台北市:五南圖書公司。
陳志偉(民96)。生醫詞彙辨識:利用隱藏式馬可夫模型。國立台灣大學醫學工程學研究所碩士論文,未出版,台北市。
陳品守,林紋正(民98)。中文健康資訊搜尋結果自動分群。JCMIT2009。國際醫學資訊研討會論文集。
陳順宇(民87)。多變量分析(2版)。台北市:華泰書局。
陳琬茹(民96)。影響醫師使用「可攜式電子病歷」意願之相關因素研究。臺北醫學大學醫務管理學研究所碩士論文,未出版,台北市。
覃業明(民90)。科技接受模型之實證研究-以國內醫療網站為例。國立成功大學資訊管理研究所碩士論文,未出版,台南市。
廖宜岑(民95)。醫療資訊入口網站使用意願之研究。中國醫藥大學醫務管理學研究所碩士論文,未出版,台中市。
Compeau, D. R., & Higgins, C. A. (1995). Computer Self-Efficacy: Development of a Measure and Initial Test. MIS Quarterly, 19(2), 189-211.
D. D. Lewis & W. B. Croft (1989). Term Clustering of Syntactic Phrases. In Proceedings of the Thirteenth Annual International ACM SIGIR Conference on Research and Development in Information Retrieval, 385-404.
Davis, F. D. (1989). Perceived Usefulness, Perceived Ease of Use, and User Acceptance of Information Technology. MIS Quarterly, 13(3), 319-340.
Davis, F. D., Bagozzi, R. P., & Warshaw, P. R. (1989). User acceptance of computer technology: A comparison of two theoretical models. Management Science, 35, 982-1003.
D. Randall Wilson, & Tony R. Martinez. (1997). Improved Heterogeneous Distance Functions. Journal of Artificial Intelligence Research, 6,1-34.
Hanisch, D., Fluck, J., Mevissen, H. & Zimmer,R. (2003). Playing biology’s name game: Identifying protein names in scientific text. Pac. Symp. Biocomput 8,403–14.
Hirschman L., Morgan AA., & Yeh AS. (2002). Rutabaga by any other name: extracting biological names. J Biomed Inform.,35(4),247-59.
Fishbein, M., & Ajzen, I. (1975). Belief, Attitude, Intention, and Behavior: An Introduction to Theory and Research. Reading, MA: Addison-Wesley.
Fukuda K., Tamura A., Tsunoda T., & Takagi T. (1998). Toward information extraction: identifying protein names from biological papers. Pac Symp Biocomput. ,707-18.
J. B. MacQueen,Some Methods for classification and Analysis of Multivariate observations. Proceedings of 5-th Berkeley Symposium on Mathematical Statistics and Probability", Berkeley, University of California Press, 1967, 1:281-297.
Liaw, S. S. (2002). Understanding user perceptions of World-wide Web environments, Journal of Computer Assisted learning, 18(2), 139-150.
Plouffe, C. R., Hulland, J.S., & Vandenbosch, M. (2001). Research report: Richness versus parsimony in modeling technology adoption decisions: Understanding merchant adoption of a smart card-based payment system. Information System Research 2001,12, 208-222.
Ramayah, T. & Aafaqi, B. ( 2004). Role of Self-Efficacy in e-Library usage among Students of a Public University in Malaysia. Malaysian Journal of Library & Information Science, Vol. 9(1),39-57.
Richard J. Holden & Ben-Tzion Karsh (2010). The Technology Acceptance Model: Its past and its future in health care. Journal of Biomedical Informatics,43(1),159-172.
Venkatesh, V., & Davis, F. D.( 2000). A Theoretical Extension of the Technology Acceptance Model: Four Longitudinal Field Studies. Management Science; 46(2), 186-204.
Witold Pedrycz. Knowledge-Based Clustering: From Data to Information Granules 2005; Jan:6-9,17.

------------------------------------------------------------------------ 第 3 筆 ---------------------------------------------------------------------
系統識別號 U0007-0108201021002400
論文名稱(中文) 醣皮質固醇與缺氧對於大腦皮質神經元細胞中Fkbp5表現調控之調節研究
論文名稱(英文) Regulation of Fkbp5 Expression by Glucocorticoid and Hypoxia in Cortical Neurons
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 歐陽婷婷
學號 M120097009
學位類別 碩士
語文別 中文
口試日期 2010-06-25
論文頁數 68頁
口試委員 指導教授-王家儀
共同指導教授-李怡萱
委員-蘇俊魁
委員-劉興政
委員-周志銘
關鍵字(中) 醣皮質固醇
缺氧
大腦皮質神經元細胞
Fkbp5
關鍵字(英) Glucocorticoid
Hypoxia
Cortical Neurons
Fkbp5
學科別分類
中文摘要 醣皮質固醇(Glucocorticoids簡稱GC)為一種類固醇荷爾蒙,在個體受到外界環境壓力時,因啟動hypothalamic-pituitary-adrenal (HPA) axis而促使其由腎上腺皮質的分泌量增加,作用在其受體Glucocorticoid receptor (GR)上,GR為一種配體活化型轉錄因子,受GC活化後,會進核影響其標的基因之表現。其中Fkbp5以及5-HT1A receptor (Htr1a)為由GR所調控且與憂鬱症相關的基因。Fkbp5的蛋白質產物為FKBP51,會與Heat shock protein 90 (HSP90)(結合形成複合體共同抑制GR的活性,形成負回饋迴路(negative feedback loop)。近來的研究顯示血清中的FKBP51含量與個體對抗憂鬱症藥物的敏感性有關,然而,FKBP51目前在憂鬱症或是環境壓力所造成的精神異常中所扮演的角色仍屬未知。在本研究中我們發現老鼠的Fkbp5 mRNA level在一天裡存在著生理節律的變化,而此種改變在血清中buffy coat及海馬迴(hippocampus)中為正向相關(positive correlation),因此推測在臨床研究中病人血清中所測得的Fkbp5 mRNA level的變化似乎可正比於其在腦中的含量變化。在本實驗中,我們使用初代培養的大腦皮質神經細胞(cortical neurons),體外培養十天使其趨近發育成熟,發現給予GC dexamethasone (Dex)處理12小時會增加Fkbp5 mRNA但會降低5-HT1A receptor基因的表現量,而Dex增加Fkbp5 mRNA的現象會被化學性缺氧藥劑:cobalt chloride (CoCl2)時所抑制。再者,長時間投予神經細胞Dex以模擬個體處於慢性壓力的情況下,不但會造成Fkbp5 mRNA表現持續增加且對抑制性 5-HT1A receptor的基因表現也有增加的趨勢,但是神經興奮所誘導的細胞內鈣濃度升高反應反而增加,而GR蛋白質表現量則是減少的。綜合以上結果,本研究發現短期及長期投予醣皮質固醇雖然皆會增加Fkbp5,但對5-HT1A receptor基因表現的影響則可能為了神經興奮性恆定的維持而有相反的結果。而缺氧壓力對於醣皮質固醇誘導Fkbp5表現的抑制作用,表示其可能會干擾Fkbp5對GR的負回饋調控。
英文摘要 Glucocorticoid receptor (GR) is a ligand-activated transcription factor, activated upon binding to glucocorticoids (GCs) when the hypothalamic-pituitary-adrenal (HPA) axis is aroused by stress. FK506 binding protein 5 (Fkbp5) and 5-HT1A receptor (Htr1a) are two of the GR-downstream genes, which were found related to depression in patients. Fkbp5 encodes FKBP51 protein chaperoned by Heat shock protein (HSP90) to form a negative feedback loop for the regulation of GR sensitivity. Recent studies revealed that the serum level of FKBP51 in patients with depression seems to associate with the sensitivity to the anti-psychotic drug treatment. However, the role of FKBP51 in the etiology of depression and the stress-related mental disorders remains unknown. In the present study, we examined the effects of GC, and in combination of hypoxic stress, on the expression of Fkbp5 and 5-HT1A receptor in primary cultured cortical neurons at 10 days-in-vitro (10 DIV). The results show that 12-h treatment with GR agonist dexamethasone (Dex), a synthetic glucocorticoid, increased the expressions of both Fkbp5 and Htr1a in primary cultured cortical neurons. The Dex-increased Fkbp5 was suppressed by cobalt chloride that induces chemical hypoxia. Interestingly, using prolonged Dex treatment (48-96 h) onto cortical neurons to simulate chronic stress in the brain elevated both Fkbp5 and Htr1a gene expression, with the Fkbp5 upregulation showing a two-phase induction profile. However, the prolonged Dex treatment showed sustained reduction of GR proteins, and increased responsiveness to the depolarizing agent (high K+)-induced intracellular calcium increase. Together, the results suggest that both acute and prolonged GC treatments upregulate Fkbp5 expression, which may negatively feedback the GR expression in cortical neurons. Hypoxia seems to interfere with this feedback mechanism by inhibiting the GC-induced Fkbp5 expression. Prolonged GC treatment reverses the acute GC repression of 5-HT1A receptor expression, which may cause the changes in the cortical excitability during chronic stress.
論文目次 中文摘要----------------------------------------------------------------I
英文摘要--------------------------------------------------------------III
前言----------------------------------------------------------------------1
實驗方法與材料-----------------------------------------------------15
結果--------------------------------------------------------------------32
討論--------------------------------------------------------------------54
結論--------------------------------------------------------------------58
參考文獻--------------------------------------------------------------59
參考文獻 Almawi, W. Y. and O. K. Melemedjian (2002). Molecular mechanisms of glucocorticoid antiproliferative effects: antagonism of transcription factor activity by glucocorticoid receptor. J Leukoc Biol 71, 1: 9-15.
Andrade, R., R. C. Malenka and R. A. Nicoll (1986). A G protein couples serotonin and GABAB receptors to the same channels in hippocampus. Science 234, 4781: 1261-5.
Bao, A. M., G. Meynen and D. F. Swaab (2008). The stress system in depression and neurodegeneration: focus on the human hypothalamus. Brain Res Rev 57, 2: 531-53.
Binder, E. B. (2009). The role of FKBP5, a co-chaperone of the glucocorticoid receptor in the pathogenesis and therapy of affective and anxiety disorders. Psychoneuroendocrinology 34 Suppl 1: S186-95.
Binder, E. B., R. G. Bradley, W. Liu, M. P. Epstein, T. C. Deveau, K. B. Mercer, Y. Tang, C. F. Gillespie, C. M. Heim, C. B. Nemeroff, A. C. Schwartz, J. F. Cubells and K. J. Ressler (2008). Association of FKBP5 polymorphisms and childhood abuse with risk of posttraumatic stress disorder symptoms in adults. JAMA 299, 11: 1291-305.
Binder, E. B., D. Salyakina, P. Lichtner, G. M. Wochnik, M. Ising, B. Putz, S. Papiol, S. Seaman, S. Lucae, M. A. Kohli, T. Nickel, H. E. Kunzel, B. Fuchs, M. Majer, A. Pfennig, N. Kern, J. Brunner, S. Modell, T. Baghai, T. Deiml, P. Zill, B. Bondy, R. Rupprecht, T. Messer, O. Kohnlein, H. Dabitz, T. Bruckl, N. Muller, H. Pfister, R. Lieb, J. C. Mueller, E. Lohmussaar, T. M. Strom, T. Bettecken, T. Meitinger, M. Uhr, T. Rein, F. Holsboer and B. Muller-Myhsok (2004). Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment. Nat Genet 36, 12: 1319-25.
Chen, Y. and N. J. Penington (1996). Differential effects of protein kinase C activation on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones. J Physiol 496 ( Pt 1): 129-37.
Clarke, W. P., M. De Vivo, S. G. Beck, S. Maayani and J. Goldfarb (1987). Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate. Brain Res 410, 2: 357-61.
Clarke, W. P., F. D. Yocca and S. Maayani (1996). Lack of 5-hydroxytryptamine1A-mediated inhibition of adenylyl cyclase in dorsal raphe of male and female rats. J Pharmacol Exp Ther 277, 3: 1259-66.
Dallman, M. F., A. M. Strack, S. F. Akana, M. J. Bradbury, E. S. Hanson, K. A. Scribner and M. Smith (1993). Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14, 4: 303-47.
Dardzinski, B. J., S. L. Smith, J. Towfighi, G. D. Williams, R. C. Vannucci and M. B. Smith (2000). Increased plasma beta-hydroxybutyrate, preserved cerebral energy metabolism, and amelioration of brain damage during neonatal hypoxia ischemia with dexamethasone pretreatment. Pediatr Res 48, 2: 248-55.
Davies, T. H., Y. M. Ning and E. R. Sanchez (2002). A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins. J Biol Chem 277, 7: 4597-600.
De Vivo, M. and S. Maayani (1986). Characterization of the 5-hydroxytryptamine1a receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J Pharmacol Exp Ther 238, 1: 248-53.
Diamond, D. M., M. C. Bennett, D. A. Engstrom, M. Fleshner and G. M. Rose (1989). Adrenalectomy reduces the threshold for hippocampal primed burst potentiation in the anesthetized rat. Brain Res 492, 1-2: 356-60.
Diaz, R., R. W. Brown and J. R. Seckl (1998). Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions. J Neurosci 18, 7: 2570-80.
Donald, R. A., C. Redekopp, V. Cameron, M. G. Nicholls, J. Bolton, J. Livesey, E. A. Espiner, J. Rivier and W. Vale (1983). The hormonal actions of corticotropin-releasing factor in sheep: effect of intravenous and intracerebroventricular injection. Endocrinology 113, 3: 866-70.
Gardner, D. S., A. J. Fletcher, A. L. Fowden and D. A. Giussani (2001). Plasma adrenocorticotropin and cortisol concentrations during acute hypoxemia after a reversible period of adverse intrauterine conditions in the ovine fetus during late gestation. Endocrinology 142, 2: 589-98.
Goldstein, R. E., A. D. Cherrington, G. W. Reed, D. B. Lacy, D. H. Wasserman and N. N. Abumrad (1994). Effects of chronic hypercortisolemia on carbohydrate metabolism during insulin deficiency. Am J Physiol 266, 4 Pt 1: E618-27.
Hackett, P. H., R. C. Roach, R. A. Wood, R. G. Foutch, R. T. Meehan, D. Rennie and W. J. Mills, Jr. (1988). Dexamethasone for prevention and treatment of acute mountain sickness. Aviat Space Environ Med 59, 10: 950-4.
Heisler, L. K., H. M. Chu, T. J. Brennan, J. A. Danao, P. Bajwa, L. H. Parsons and L. H. Tecott (1998). Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Acad Sci U S A 95, 25: 15049-54.
Hensler, J. G. (2003). Regulation of 5-HT1A receptor function in brain following agonist or antidepressant administration. Life Sci 72, 15: 1665-82.
Hensler, J. G., G. B. Kovachich and A. Frazer (1991). A quantitative autoradiographic study of serotonin1A receptor regulation. Effect of 5,7-dihydroxytryptamine and antidepressant treatments. Neuropsychopharmacology 4, 2: 131-44.
Hoyer, D., D. E. Clarke, J. R. Fozard, P. R. Hartig, G. R. Martin, E. J. Mylecharane, P. R. Saxena and P. P. Humphrey (1994). International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 46, 2: 157-203.
Hubler, T. R. and J. G. Scammell (2004). Intronic hormone response elements mediate regulation of FKBP5 by progestins and glucocorticoids. Cell Stress Chaperones 9, 3: 243-52.
Innis, R. B. and G. K. Aghajanian (1987). Pertussis toxin blocks 5-HT1A and GABAB receptor-mediated inhibition of serotonergic neurons. Eur J Pharmacol 143, 2: 195-204.
Ising, M., A. M. Depping, A. Siebertz, S. Lucae, P. G. Unschuld, S. Kloiber, S. Horstmann, M. Uhr, B. Muller-Myhsok and F. Holsboer (2008). Polymorphisms in the FKBP5 gene region modulate recovery from psychosocial stress in healthy controls. Eur J Neurosci 28, 2: 389-98.
Joels, M. and E. R. de Kloet (1992). Control of neuronal excitability by corticosteroid hormones. Trends Neurosci 15, 1: 25-30.
Johnson, R. G., D. Fiorella, J. C. Winter and R. A. Rabin (1997). [3H]8-OH-DPAT labels a 5-HT site coupled to inhibition of phosphoinositide hydrolysis in the dorsal raphe. Eur J Pharmacol 329, 1: 99-106.
Johnson, T. S., P. B. Rock, C. S. Fulco, L. A. Trad, R. F. Spark and J. T. Maher (1984). Prevention of acute mountain sickness by dexamethasone. N Engl J Med 310, 11: 683-6.
Kadekaro, M., M. Ito and P. M. Gross (1988). Local cerebral glucose utilization is increased in acutely adrenalectomized rats. Neuroendocrinology 47, 4: 329-34.
Kerr, D. S., L. W. Campbell, M. D. Applegate, A. Brodish and P. W. Landfield (1991). Chronic stress-induced acceleration of electrophysiologic and morphometric biomarkers of hippocampal aging. J Neurosci 11, 5: 1316-24.
Kerr, D. S., L. W. Campbell, O. Thibault and P. W. Landfield (1992). Hippocampal glucocorticoid receptor activation enhances voltage-dependent Ca2+ conductances: relevance to brain aging. Proc Natl Acad Sci U S A 89, 18: 8527-31.
Kia, H. K., M. C. Miquel, M. J. Brisorgueil, G. Daval, M. Riad, S. El Mestikawy, M. Hamon and D. Verge (1996). Immunocytochemical localization of serotonin1A receptors in the rat central nervous system. J Comp Neurol 365, 2: 289-305.
Kodama, T., N. Shimizu, N. Yoshikawa, Y. Makino, R. Ouchida, K. Okamoto, T. Hisada, H. Nakamura, C. Morimoto and H. Tanaka (2003). Role of the glucocorticoid receptor for regulation of hypoxia-dependent gene expression. J Biol Chem 278, 35: 33384-91.
Lee, M. K., S. N. Graham and P. E. Gold (1988). Memory enhancement with posttraining intraventricular glucose injections in rats. Behav Neurosci 102, 4: 591-5.
Lopez, J. F., D. T. Chalmers, K. Y. Little and S. J. Watson (1998). A.E. Bennett Research Award. Regulation of serotonin1A, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for the neurobiology of depression. Biol Psychiatry 43, 8: 547-73.
Martini, L. and C. Monpurgo (1955). Neurohumoral control of the release of adrenocorticotrophic hormone. Nature 175, 4469: 1127-8.
Meaney, M. J., D. H. Aitken, C. van Berkel, S. Bhatnagar and R. M. Sapolsky (1988). Effect of neonatal handling on age-related impairments associated with the hippocampus. Science 239, 4841 Pt 1: 766-8.
Meijer, O. C. and E. R. de Kloet (1998). Corticosterone and serotonergic neurotransmission in the hippocampus: functional implications of central corticosteroid receptor diversity. Crit Rev Neurobiol 12, 1-2: 1-20.
Morishima, Y., P. J. Murphy, D. P. Li, E. R. Sanchez and W. B. Pratt (2000). Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket. J Biol Chem 275, 24: 18054-60.
Ou, X. M., J. M. Storring, N. Kushwaha and P. R. Albert (2001). Heterodimerization of mineralocorticoid and glucocorticoid receptors at a novel negative response element of the 5-HT1A receptor gene. J Biol Chem 276, 17: 14299-307.
Pariante, C. M. and A. H. Miller (2001). Glucocorticoid receptors in major depression: relevance to pathophysiology and treatment. Biol Psychiatry 49, 5: 391-404.
Parks, C. L., P. S. Robinson, E. Sibille, T. Shenk and M. Toth (1998). Increased anxiety of mice lacking the serotonin1A receptor. Proc Natl Acad Sci U S A 95, 18: 10734-9.
Penington, N. J. and J. S. Kelly (1990). Serotonin receptor activation reduces calcium current in an acutely dissociated adult central neuron. Neuron 4, 5: 751-8.
Penington, N. J., J. S. Kelly and A. P. Fox (1993). Whole-cell recordings of inwardly rectifying K+ currents activated by 5-HT1A receptors on dorsal raphe neurones of the adult rat. J Physiol 469: 387-405.
Pineyro, G. and P. Blier (1999). Autoregulation of serotonin neurons: role in antidepressant drug action. Pharmacol Rev 51, 3: 533-91.
Ramboz, S., R. Oosting, D. A. Amara, H. F. Kung, P. Blier, M. Mendelsohn, J. J. Mann, D. Brunner and R. Hen (1998). Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci U S A 95, 24: 14476-81.
Rhen, T. and J. A. Cidlowski (2005). Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med 353, 16: 1711-23.
Richer, M., R. Hen and P. Blier (2002). Modification of serotonin neuron properties in mice lacking 5-HT1A receptors. Eur J Pharmacol 435, 2-3: 195-203.
Rivier, C., M. Brownstein, J. Spiess, J. Rivier and W. Vale (1982). In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, beta-endorphin, and corticosterone. Endocrinology 110, 1: 272-8.
Sambhi, M. P., M. H. Weil and V. N. Udhoji (1965). Acute Pharmacodynamic Effects of Glucocorticoids; Cardiac Output and Related Hemodynamic Changes in Normal Subjects and Patients in Shock. Circulation 31: 523-30.
Sapolsky, R. M. (1996). Stress, Glucocorticoids, and Damage to the Nervous System: The Current State of Confusion. Stress 1, 1: 1-19.
Sapolsky, R. M., L. C. Krey and B. S. McEwen (1985). Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging. J Neurosci 5, 5: 1222-7.
Sapolsky, R. M., L. M. Romero and A. U. Munck (2000). How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21, 1: 55-89.
Scammell, J. G., W. B. Denny, D. L. Valentine and D. F. Smith (2001). Overexpression of the FK506-binding immunophilin FKBP51 is the common cause of glucocorticoid resistance in three New World primates. Gen Comp Endocrinol 124, 2: 152-65.
Schiene-Fischer, C. and C. Yu (2001). Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett 495, 1-2: 1-6.
Schoneveld, O. J., I. C. Gaemers and W. H. Lamers (2004). Mechanisms of glucocorticoid signalling. Biochim Biophys Acta 1680, 2: 114-28.
Smoak, K. A. and J. A. Cidlowski (2004). Mechanisms of glucocorticoid receptor signaling during inflammation. Mech Ageing Dev 125, 10-11: 697-706.
Snell, K. (1991). Regulation of hepatic glucose metabolism by insulin and counter-regulatory hormones. Proc Nutr Soc 50, 3: 567-75.
Stockmann, C. and J. Fandrey (2006). Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression. Clin Exp Pharmacol Physiol 33, 10: 968-79.
Strack, A. M., R. J. Sebastian, M. W. Schwartz and M. F. Dallman (1995). Glucocorticoids and insulin: reciprocal signals for energy balance. Am J Physiol 268, 1 Pt 2: R142-9.
Tatro, E. T., I. P. Everall, M. Kaul and C. L. Achim (2009). Modulation of glucocorticoid receptor nuclear translocation in neurons by immunophilins FKBP51 and FKBP52: implications for major depressive disorder. Brain Res 1286: 1-12.
Tomas, F. M., H. N. Munro and V. R. Young (1979). Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of N tau-methylhistidine. Biochem J 178, 1: 139-46.
Van de Kar, L. D. (1991). Neuroendocrine pharmacology of serotonergic (5-HT) neurons. Annu Rev Pharmacol Toxicol 31: 289-320.
Vermeer, H., B. I. Hendriks-Stegeman, B. van der Burg, S. C. van Buul-Offers and M. Jansen (2003). Glucocorticoid-induced increase in lymphocytic FKBP51 messenger ribonucleic acid expression: a potential marker for glucocorticoid sensitivity, potency, and bioavailability. J Clin Endocrinol Metab 88, 1: 277-84.
Yehuda, R., G. Cai, J. A. Golier, C. Sarapas, S. Galea, M. Ising, T. Rein, J. Schmeidler, B. Muller-Myhsok, F. Holsboer and J. D. Buxbaum (2009). Gene expression patterns associated with posttraumatic stress disorder following exposure to the World Trade Center attacks. Biol Psychiatry 66, 7: 708-11.
Yehuda, R., J. A. Golier, R. K. Yang and L. Tischler (2004). Enhanced sensitivity to glucocorticoids in peripheral mononuclear leukocytes in posttraumatic stress disorder. Biol Psychiatry 55, 11: 1110-6.

------------------------------------------------------------------------ 第 4 筆 ---------------------------------------------------------------------
系統識別號 U0007-0108201022523700
論文名稱(中文) 桑黃萃取物Hispolon抑制腦部微膠細胞發炎反應之機制探討
論文名稱(英文) Inhibitory mechanism of Hispolon on the inflammatory responses of microglia cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 林青蓓
學號 M120097021
學位類別 碩士
語文別 中文
口試日期 2010-07-05
論文頁數 82頁
口試委員 指導教授-陳彥州
委員-李哲夫
委員-蕭哲志
關鍵字(中) 桑黃
Hispolon
微膠細胞
腦部發炎
血紅素氧化酵素-1
關鍵字(英) Phellinus linteus
Hispolon
microglia
brain inflammation
HO-1
學科別分類
中文摘要 微膠細胞在調控神經發炎反應上扮演重要的角色,它們為各種神經退化性疾病病理發展的中心關鍵要角。因此,可以調節微膠細胞活化狀態的化合物可能具有治療神經退化性疾病的潛力。先前有研究指出,從桑黃中所分離出的Hispolon具有抗腫瘤的活性,然而, Hispolon (HIS)的抗發炎活性主要透過的作用分子標的以及作用機制仍然不清楚。本實驗探討了HIS對脂多醣(LPS)及脂磷壁酸(LTA)誘導微膠細胞株BV-2之iNOS/NO產生的影響,本研究成果發現了HIS能有效抑制LPS及LTA誘導的NO產生且此效果具劑量依存性;同時,在HIS處理下,LPS及LTA誘導iNOS蛋白表現亦顯著被抑制。此外,HIS的給予會造成BV-2細胞株血紅素氧化酵素-1 (HO-1)蛋白表現量的增加,且經由各種Kinase抑制劑證實了HIS誘導HO-1蛋白表現會被ERK抑制劑PD98059所抑制,這結果指出在BV-2細胞中,ERK活化參與了HIS所誘導之HO-1蛋白表現。
此外,LPS及LTA處理之BV-2中,JNK抑制劑SP600125能有效抑制iNOS/NO之產生,結果同樣也發現HIS可抑制LPS及LTA誘導JNK磷酸化並抑制下游轉錄因子NF-kB ,c-Jun的活化最後造成NO生成及iNOS蛋白表現減少;進一步我們發現LPS及LTA能誘導BV-2細胞走向細胞凋亡,此細胞凋亡的結果能被NOS抑制劑L-NAME及HIS所抑制。此外,以HO-1抑制劑SnPP處理下, HIS抑制LPS及LTA誘導NO生成之活性則顯著降低。
因此本研究結果證實了HIS能經由誘導HO-1蛋白表現來抑制LPS及LTA誘導NO產生而達到保護BV-2細胞免於其受LPS及LTA誘導之細胞凋亡。因此, HIS或許可以藉由減輕在各種神經退化性疾病的病程進展過程中的微膠細胞過度發炎狀態而作為一個有潛能的治療藥物。
英文摘要 Elevation of Nitric Oxide (NO) production via stimulating inducible nitric oxide synthase (iNOS) has been shown in activated microglia cells during brain inflammation.Therefore, agents with ability to reduce iNOS/NO production in activated microglia cells possess beneficial effects in the treatment of several neurodegenerative diseases.Hispolon (HIS), a compound derived from Phellinu linteus, has been shown to possess several beneficial effects such as antioxidant, inhibition of tumor growth and metastasis. The anti-inflammatory effects of HIS in microglia,however, remains unclear.
In the present study, HIS exhibited concentration-dependent inhibition of LPS/LTA-induced iNOS protein expression/NO production accompanied by Heme Oxygenase-1 (HO-1) induction in microglia cells BV-2. Also, time and concentration-dependent induction of HO-1 protein by HIS was identified in BV-2 cells. Application of ERK inhibitor U0126 reduced HO-1 protein expression elicited by HIS, indicating ERK activation participated in HIS-induced HO-1 protein expression. Additionally, LPS/LTA-induced JNK phosphorylation was suppressed by adding HIS in BV-2 cells. Also, inhibition of LPS/LTA-induced NF-kB P65 nuclear translocation, I?羠 protein degradation and c-Jun protein expression in BV-2 cells under HIS treatment were observed. Furthermore, HIS and NOS inhibitor significantly protected against LPS /LTA-induced apoptosis, characterized by DNA fragmentation assay and Caspase 9 activation. Moreover, inhibitory effect of HIS on LPS/LTA-induced NO production was attenuated by addition of HO-1 inhibitor SnPP, suggesting that HO-1 induction in part participated in anti-inflammatory actions of HIS.

In conclusion, data of the present study provided evidence supporting the inhibitory effects of HIS on LPS/LTA-induced inflammatory responses in microglia cells BV-2.Furthermore, beneficial effects of HIS in protection of BV-2 cells from apoptosis via reducing LPS/ LTA-induced iNOS protein expression/NO production were also demonstrated.
論文目次 目錄
目錄…………………………………………………………………… i
圖目錄………………………………………………………………… iv
縮寫表...................................................1
中文摘要.................................................................................................. 2
Abstract.................................................................................................... 4
I、前言.....................................................................................................6
II、實驗材料…………………………………………………………. 15
II、實驗材料…………………………………………………………. 16
II-1、材料…………………………………………………………….. 16
II-2、藥品試劑……………………………………………………….. 17
II-3、細胞株及培養液……………………………………………….. 19
II-4、抗體試劑……………………………………………………….. 19
II-5、一般儀器……………………………………………………….. 20
III、實驗方法………………………………………………………… 22
III、實驗方法………………………………………………………… 23
III-1、藥品試劑之配製………………………………………………. 23
III-2、細胞培養方法…………………………………………………. 25
III-3、MTT細胞毒性之試驗………………………………………....26

III-4、細胞蛋白質抽取………………………………………………. 27
III-5、西方點墨法分析………………………………………………. 28
III-6、統計資料………………………………………………………. 28
IV、實驗結果………………………………………………………… 29
IV-1、Hispolon對微膠細胞BV-2之毒性影響 …………………….30
IV-2、Hispolon抑制酯多醣(LPS)與脂磷壁酸(LTA)活化誘導型一氧化氮合成酵素(iNOS)及一氧化氮(NO)生成. …………………………..30
IV-3、Hispolon可誘導HO-1基因表現……………………………. .32
IV-4、Hispolon透過活化ERK (Extracellular signal-regulated kinase)
路徑來誘導HO-1蛋白表現………………………………………..... 33
IV-5、Hispolon經由增加ROS產量來誘導HO-1蛋白表現……... .34
IV-6、Hispolon抑制酯多醣(LPS)與脂磷壁酸(LTA)誘導iNOS表現及NO生成主要透過活化HO-1蛋白表現…………………………….. 34
IV-7、HO-1抑制劑tin protoporphyrin (SnPP)減弱Hispolon抑制LPS及LTA誘導NO生成之作用………………………………………... 35
IV-8、Hispolon抑制酯多醣(LPS)與脂磷壁酸(LTA)誘導JNK磷酸化
………………………………………………………………………....36
IV-9、Hispolon可抑制酯多醣(LPS)與脂磷壁酸(LTA)誘導轉錄因子
NF-?羠之活化…………………………………………………………38

IV-10、Hispolon可抑制酯多醣(LPS)與脂磷壁酸(LTA)誘導轉錄因子
AP-1之活化…………………………………………………………. 39
IV-11、Hispolon透過抑制iNOS蛋白表現進而減少NO生成而非直接清除NO …………………………………………………………….. .39
IV-12、Hispolon具抑制酯多醣(LPS)與脂磷壁酸(LTA)誘導之細胞凋亡的作用來自於其抑制NO生成的活性…………………………… ...40
IV-13、Hispolon及其合成結構類似物的抗發炎以及細胞保護活性之比較…………………………………………………………………...42
V、討論………………………………………………………………44
VI、參考文獻……………………………………………………….. 51

















圖目錄

Fig1. Effect of HIS on the viability of BV-2 cells……………………... 63
Fig2. HIS inhibition of LPS/LTA-induced iNOS /NO production with concurrent induction of HO-1 protein expression in BV-2 cells………. 64
Fig3. HIS induction of HO-1 protein expression in BV-2 cells………...66
Fig4. Involvement of ERK protein phosphorylation in the HIS-induced
HO-1 protein expression………………………………………………. 67
Fig5. Contribution of ROS production in the HIS-induced HO-1 protein expression……………………………………………………………... 68
Fig6. HIS inhibition of LPS/LTA-induced iNOS /NO production via enhancing HO-1 protein expression…………………………………... 69
Fig7. Tin protoporphyrin (SnPP) attenuation of the inhibitory effects of HIS on LPS/LTA-induced NO production……………………………. 70
Fig8. JNK involved in the LPS/LTA-induced iNOS /NO production in BV-2 cells……………………………………………………………... 71
Fig9. HIS inhibition of LPS/LTA-induced JNK activation …………....72
Fig10. NF-kB involved in LPS-induced iNOS/NO production in BV-2 cells……………………………………………………………………..73
Fig11. HIS inhibition of LPS/LTA-induced p65 nuclear translocation and IkB protein degradation…………………………………………….. ....74
Fig12. HIS inhibition of LPS-induced c-Jun protein expression…….... 75
Fig13. No direct inhibitory effects of HIS on Nitric Oxide…………......76
Fig14. HIS attenuation of LPS/LTA-induced apoptosis via inhibition of LPS/LTA-induced NO production ……………………………………...77
Fig15. Structure-Activity Relationship for the antiinflammatory and antiapoptotic effect of Hispolon and its analogue compounds………..79
附錄一……………………………………………………………… ...82
參考文獻 Alcaraz MJ, Isabel D, Maria L F, Isabel G, José M C (2005) Potential role of heme oxygenase-1 in the progression of rat adjuvant arthritis. Lab Invest 85, 34–44
Aloisi F (1999) The role of microglia and astrocytes in CNS immune surveillance and immunopathology. Adv Exp Med Biol 468:123–133
Awadh Ali, N. A., R. A. Mothana (2003) Antiviral activity of Inonotus hispidus. Fitoterapia 74(5): 483-485
Belcher JD, Mahaseth H, Welch TE, Otterbein LE, Hebbel RP,Vercellotti GM (2006) Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice. J Clin Invest 116: 808–816
Boje KM and Arora PK(1992) Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Res 587:250–256
Brown DR (2001) Microglia and prion disease. Microsc Res Tech 54:71–80
Chang, H. Y., W. H. Peng (2007) Hepatoprotective and Antioxidant Effects of Ethanol Extract from Phellinus merrillii on carbon tetrachloride-induced liver damage. Am J Chin Med 35(5): 793-804
Chang, H. Y., M. J. Sheu (2009) Analgesic Effects and the Mechanisms of Anti-Inflammation of Hispolon in Mice." Evid Based Complement Alternat Med
Chao CC, Hu S, Molitor TW, Shaskan EG, and Peterson PK (1992) Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. J Immunol 149:2736–2741
Chen YC, Shen SC, Lee WR, Lin HY, Ko CH, Lee TJF(2002) Nitric oxide and prostaglandin E2 participate in lipopolysaccharide/interferon-??-induced heme oxygenase 1 and prevent RAW264.7 macrophages from UV-irradiation-induced cell death. J Cell. Biochem. 86:331-9
Chen, W., F. Y. He (2006) The apoptosis effect of hispolon from Phellinus linteus (Berkeley & Curtis) Teng on human epidermoid KB cells." J Ethnopharmacol 105(1-2): 280-285
Chen, W., Z. Zhao (2008) Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway." Free Radic Biol Med 45(1): 60-72
Chung SW, Chen YH, Perrella MA (2005) Role of Ets-2 in the regulation of heme oxygenase-1 by endotoxin. J Biol Chem 280:4578–4584
Chung SW, Chen YH, Yet SF, Layne MD, Perrella MA (2006) Endotoxin-induced down-regulation of Elk-3 facilitates heme oxygenase-1 induction in macrophages. J Immunol 176: 2414–2420
Dickson DW, Lee SC, Mattiace LA, Yen SH, and Brosnan C (1993) Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. Glia 7:75–83
Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. (2001) Heme oxygenase-1 protects against vascular constriction and proliferation. Nat. Med. 7:693-98
Gao PS, Kawada H, Kasamatsu T, Mao XQ, Roberts MH, Miyamoto Y, Yoshimura M, Saitoh Y, Yasue H, Nakao K, Adra CN, Kun JF, Moro-oka S, Inoko H, Ho LP, Shirakawa T, Hopkin JM (2000) Variants of NOS1, NOS2, and NOS3 genes in asthmatics. Biochem. Biophys. Res. Commun. 267:761-3
Hawiger J (2001) Innate immunity and inflammation: a transcriptional paradigm. Immunol. Res. 23:99-109
Hock TD, Liby K, Wright MM, McConnell S, Schorpp-Kistner M,Ryan TM (2007) JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells. J Biol Chem 282: 6875–6886
Kaizaki A, Tanaka S, Ishige K, Numazawa S, Yoshida T (2006) The neuroprotective effect of heme oxygenase (HO) on oxidative stress in HO-1 siRNA-transfected HT22 cells. Brain Res 1108: 39–44
Kallin A, Johannessen LE, Cani PD, Marbehant CY, Essaghir A,Foufelle F (2007) SREBP-1 regulates the expression of hemeoxygenase 1 and the phosphatidylinositol-3 kinase regulatorysubunit p55g. J Lipid Res 48: 1628–1636
Katsuoka F, Motohashi H, Ishii T, Aburatani H, Engel JD, Yamamoto M (2005) Genetic evidence that small maf proteins are essential for the activation of antioxidant response element-dependent genes. Mol Cell Biol 25: 8044–8051
Kronke G, Kadl A, Ikonomu E, Bluml S, Furnkranz A, Sarembock IJ (2007) Expression of heme oxygenase-1 in human vascularcells is regulated by peroxisome proliferator-activated receptors. Arterioscler Thromb Vasc Biol 27: 1276–1282
Lin CM, Chen CT, Lee HH, Lin JK (2002) Prevention of cellular ROS damage by isovitexin and related flavonoids. Planta Medica 68:365-7
Lee, I. K. and B. S. Yun (2007) Highly oxygenated and unsaturated metabolites providing a diversity of hispidin class antioxidants in the medicinal mushrooms Inonotus and Phellinus." Bioorg Med Chem 15(10): 3309-3314
Liu B and Hong JS (2003) Role of Microglia in Inflammation-Mediated Neurodegenerative Diseases: Mechanisms and Strategies for Therapeutic Intervention. J Pharmacol Exp Ther. 304(1):1-7
Lu, T. L., G. J. Huang (2009) Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited ERK1/2 activity in breast and bladder cancer cells." Food Chem Toxicol 47(8): 2013-2021
Lu, T. L., G. J. Huang (2010) Hispolon Promotes MDM2 Downregulation through Chaperone-Mediated Autophagy. Biochem Biophys Res Commun.
Marletta MA (1993) Nitric oxide synthase structure and mechanism. J. Biol. Chem. 268:12231-4
McGeer PL, Itagaki S, Boyes BE, and McGeer EG (1988) Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38:1285–1291
McGuire SO, Ling ZD, Lipton JW, Sortwell CE, Collier TJ, and Carvey PM (2001) Tumor necrosis factor alpha is toxic to embryonic mesencephalic dopamine neurons. Exp Neurol 169:219–230
Mo, S., S. Wang (2004) Phelligridins C-F: cytotoxic pyrano[4,3-c][2]benzopyran-1,6-dione and furo[3,2-c]pyran-4-one derivatives from the fungus Phellinus igniarius. J Nat Prod 67(5): 823-828.
Moncada S, Bolanos JP (2006) Nitric oxide, cell bioenergetics and neurodegeneration. J Neurochem 97(6):1676-89
Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk M, Davis RJ, Flavell RA, Choi AM (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat. Med. 6:422-28
PJ Syapin (2008) Regulation of haeme oxygenase-1 for treatment of neuroinflammation and brain disorders Brit J Pharmacol 155, 623–640
Parhizgar SS (2007) Role of hemeoxygenase-1 in the inhibition of the iNOS expression by nonsteroidal anti-inflammatory drugs in C6 glioma cells. Texas Tech University Health Sciences
Park SW, Yi JH, Miranpuri G, Satriotomo I, Bowen K, Resnick DK (2007) Thiazolidinedione class of peroxisome proliferatoractivated receptor gamma agonists prevents neuronal damage, motor dysfunction, myelin loss, neuropathic pain, and inflammation after spinal cord injury in adult rats. J Pharmacol Exp Ther 320
Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD (2006) Blood–brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 1: 223–236
Piehl F, Lidman O (2001) Neuroinflammation in the rat—CNS cells and their role in the regulation of immune reactions. Immunol Rev 184: 212–225
Polazzi E, Contestabile A (2002) Reciprocal interactions between microglia and neurons: from survival to neuropathology. Rev Neurosci 13: 221–242
Raine CS (1994) Multiple sclerosis: immune system molecule expression in the central nervous system. J Neuropathol Exp Neurol 53:328–337
Ravindran, J., G. V. Subbaraju (2010) Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro. Biochem Pharmacol 79(11): 1658-1666
Regan RF, Guo Y, Kumar N (2000) Heme oxygenase-1 induction protects murine cortical astrocytes from hemoglobin toxicity. Neurosci Lett 282: 1
Reiling N, Ulmer AJ, Duchrow M, Ernst M, Flad HD, Hauschildt S. (1994) Nitric oxide synthase: mRNA expression of different isoforms in human monocytes/macrophages. Eur. J. Immunol. 24:1941-4
Rogers J, Luber-Narod J, Styren SD, and Civin WH (1988) Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer’s disease. Neurobiol Aging 9:339–349
Ryter SW, Otterbein LE, Morse D, Choi AM (2002) Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol. Cell. Biol. 234-235: 249-63
Ryter SW, Xi S, Hartsfield CL, Choi AM (2002) Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells. Antioxid. Redox Signal. 4:587-92
Ryter SW, Alam J, Choi AM (2006) Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. PhysiolRev 86: 583–650
Satoh T, Baba M, Nakatsuka D, Ishikawa Y, Aburatani H, Furuta K et al. (2003) Role of heme oxygenase-1 protein in the neuroprotective effects of cyclopentenone prostaglandin derivatives under oxidative stress. Eur J Neurosci 17: 2249–2255
Scapagnini G, Butterfield DA, Colombrita C, Sultana R, Pascale A, Calabrese V (2004) Ethyl ferulate, a lipophilic polyphenol, induces HO-1 and protects rat neurons against oxidative stress. Antioxid Redox Signal 6: 811–818
Schipper HM, Bennett DA, Liberman A, Bienias JL, Schneider JA, Kelly J (2006) Glial heme oxygenase-1 expression in Alzheimer disease and mild cognitive impairment. Neurobiol Aging 27: 252–261
Schipper HM, Cisse S, Stopa EG (1995) Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann Neurol 37: 758–768
Schipper HM, Liberman A, Stopa EG (1998) Neural heme oxygenase-1 expression in idiopathic Parkinson’s disease. Exp Neurol 150: 60–68
Schluesener HJ, Seid K, Deininger M, Schwab J (2001b) Transient in vivo activation of rat brain macrophages/microglial cells and astrocytes by immunostimulatory multiple CpG oligonucleotides. J Neuroimmunol 113: 89–94
Schmidt J, Mertz K, Morgan JI (1999) Regulation of heme oxygenase-1 expression by dopamine in cultured C6 glioma and primary astrocytes. Brain Res Mol Brain Res 73: 50–59
Seatter SC, Li MH, Bubrick MP, West MA (1995) Endotoxin pretreatment of human monocytes alters subsequent endotoxin-triggered release of inflammatory mediators. Shock 3:252-8
Shibahara S, Kitamuro T, Takahashi K (2002) Heme degradation and human disease: diversity is the soul of life. Antioxid. Redox Signal. 4:593-602
Shen SC, Lee WR, Lin HY, Huang HC, Ko CH, Yang LL, Chen YC (2002) In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E2 productions. Eur. J. Pharmacol. 446:187-94
Shibahara S, Kitamuro T, Takahashi K (2002) Heme degradation and human disease: diversity is the soul of life. Antioxid. Redox Signal. 4:593-602
Smith A, Alam J, Escriba PV, Morgan WT (1993) Regulation of heme oxygenase and metallothionein gene expression by the heme analogs, cobalt-, and tin-protoporphyrin. J. Biol. Chem. 268:7365-71
Takahashi M, Dore S, Ferris CD, Tomita T, Sawa A, Wolosker H (2000) Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer’s disease. Neuron 28: 461–473
Tamion F, Richard V, Renet S, Thuillez C (2006) Protective effects of heme-oxygenase expression against endotoxic shock: inhibitionof tumor necrosis factor-a and augmentation of interleukin-10.J Trauma 61: 1078–1084
Velichkova M, Hasson T (2005) Keap1 regulates the oxidationsensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism. Mol Cell Biol 25: 4501–4513
Vincent SR, Das S, Maines MD (1994) Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons. Neuroscience 63: 223–231
Willis D, Moore AR, Frederick R, Willoughby DA (1996) Hemeoxygenase: a novel target for the modulation of the inflammatory response. Nat Med 2: 87–90
Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin. Invest. 103:129-35
Yasui Y, Nakamura M, Onda T, Uehara T, Murata S, Matsui N (2007) Heme oxygenase-1 inhibits cytokine production by activated mast cells. Biochem Biophys Res Commun 354: 485–490
Ye X, Liu SF (2001) Lipopolysaccharide regulates constitutive and inducible transcription factor activities differentially in vivo in the rat. Biochem. Biophys. Res. Commun. 288:927-32
Yi JH, Hazell AS (2005) N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury. Brain Res 1033: 13–19
Yoshida T, Biro P, Cohen T, Muller RM, Shibahara S (1988) Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur JCBiochem 171: 457–461

------------------------------------------------------------------------ 第 5 筆 ---------------------------------------------------------------------
系統識別號 U0007-0202201000020100
論文名稱(中文) 經不同條件製備之多孔纖維支架對PC12細胞增生,分化及相關基因表現之影響
論文名稱(英文) The effect of poro-fibrous scaffolds fabricated at different conditions on PC12 cell proliferation, differentiation and related gene expressions
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 1
出版年 99
研究生(中文) 楊織萍
學號 M120096027
學位類別 碩士
語文別 中文
口試日期 2010-01-12
論文頁數 92頁
口試委員 指導教授-曾厚
委員-林峰輝
委員-張淑芬
關鍵字(中) 電氣紡絲;細胞支架;組織工程;PC12 cell;基因表現
關鍵字(英) electrospinning;scaffold;tissue engineering;PC12 cell;gene expression
學科別分類
中文摘要 神經系統受損後,很難自我再生,治療後,受損組織間常形成一個無血管供應及支持性基質的間隙,導致神經組織功能性的修復不易。本研究利用纖維組成的3D高孔洞網狀結構薄膜環境模擬細胞外間質,並利用類神經細胞:PC12細胞株為研究模式。研究主要以聚左乳酸(PLLA)與聚丁烯琥珀酸己二酸共聚物(PBSA) 製成一系列仿細胞外間質的絲狀薄膜,並以PC12細胞株為對象,將PC12細胞株培養在不同薄膜上,來探討PC12細胞株在不同薄膜上的貼附、增生情形。結果顯示細PLLA對於細胞貼附與增生均具較佳,細胞增生部分甚至比TCPS高於2.3倍,然而這都是以細胞現行表現探討所營造的環境是否利於細胞生長,無法知道細胞長遠的表現為何,因此本研究再利用基因表現的方式來探討細胞潛力。結果得知, 細PBSA有助於PC12細胞神經軸突生長,以細胞基因表現來看,由代表神經軸突錐生長的gene:GAP43及代表神經軸突生長的gene:MAP2可知,細胞培養在細PBSA於GAP43與MAP2表現上比TCPS分別高出19倍及1.7倍,其他披覆r-PGA、collagen等的薄膜效果亦相若。總結,本研究結果推論細PLLA及細PBSA較相似於細胞間質的結構,故在細胞貼附、增生及神經軸突生長較2D好,其提供促進神經細胞間的交互作用及神經細胞生長之良好環境,此對於神經再生組織工程中提供一個極佳的方向。
英文摘要 After the neuron system injured, it is hard to regenerate. There will be a gap that has no blood vessel and matrix. Therefore, the regeneration of neuron system is hard to do. I fabricate the membranes of 3D structure to mimic the extra cellular matrix, and use the quasi-neuron cell: PC12 cell lines to be the cell model for my research. PBSA and PLLA of polymers are fabricated the fibrous membranes to mimic extra cellular matrix, and seeded PC12 cell line on the differential membranes. Then, confer PC12 cell proliferation and adhesion. The results exhibit that the fine fibrous PLLA membranes improve cell adhesion and proliferation rather than TCPS to 2.3 folds. But it is the external expression of cell, and we do not know the internal expression of cell. Therefore, keep the gene expression to research the internal expression of cell. The result exhibit that the fine fibrous PLLA membranes improve the growth of neurite outgrowth cone and neurite outgrowth, that show from the GAP43 and MAP2 gene expression rather than TCPS to 19 and 17 folds. And the results of membranes are coated collagen and r-PGA are similar of non-coating membranes. Conclusion, the structure of fine fibrous PLLA and PBSA membranes are similar of the extra cellular matrix, so they improve PC12 cell proliferation, adhesion and the growth of neurite outgrowth rather than 2D structure (ex: TCPS). The fibrous PLLA and PBSA membranes can provide a good environment to improve the interaction and growth of neuron cells that provide a good approach for the nerve regeneration of tissue engineering.
論文目次 目 錄

誌謝 I
摘要 III
Abstract V
縮寫表 VII
目錄 VIII
第一章 緒論 1
1-1 前言 1
1-2 研究動機 3
1-3 研究目的 4
第二章 文獻回顧 5
2-1組織工程 5
2-2電氣紡絲 6
2-3周邊神經系統傷害與修復 7
2-4生物支架與細胞外間質 9
第三章 材料與方法 13
3-1 材料與試劑 13
3-2 儀器設備 14
3-3 研究方法 16
3-3-1 材料物化性質分析 17
3-3-2 材料滅菌 19
3-4 實驗流程圖 20
3-5 實驗步驟 21
3-5-1 薄膜製備 21
3-5-2細胞培養與薄膜檢測 23
3-5-3薄膜上細胞基因表現分析 26
第四章 結果與討論 31
4-1 纖維薄膜型態及其物理特性 32
4-1-1 纖維薄膜之製備 33
4-1-2 纖維薄膜之細胞毒性測試 35
4-1-3 纖維薄膜之親疏水性測定 37
4-2 纖維薄膜之細胞貼附試驗 38
4-3 纖維薄膜之細胞增生試驗 41
4-4 纖維薄膜對細胞活性檢測劑呈色劑吸收之
preliminary study 43
4-5 細胞電穿孔基因轉殖及細胞基因轉殖後於薄膜上生長之觀察 45
4-6 PC12細胞株培養於纖維薄膜上之基因表現 46
第五章 結論 56
第六章 未來研究方向 58
參考文獻 59
附錄 實驗數據與圖 62

參考文獻 1. D.E. Ingber, V.C. Mow, D. Butler, L. Niklason, J. Huard, J. Mao, I. Yannas, D. Kaplan, G. Vunjak-Novakovic, Tissue engineering and developmental biology: Going biomimetic. Tissue Engineering, 2006. 12(12): p. 3265-3283.
2. X. Zong, K. Kim, D. Fang, S. Ran, B.S. Hsiao, B. Chu, Structure and process relationship of electrospun bioabsorbable nanofiber membranes. Polymer, 2002. 43(16): p. 4403-4412.
3. D.H. Reneker, I. Chun, Nanometre diameter fibres of polymer, produced by electrospinning. Nanotechnology, 1996. 7(3): p. 216-223.
4. W.J. Li, C.T. Laurencin, E.J. Caterson, R.S. Tuan, F.K. Ko, Electrospun nanofibrous structure: A novel scaffold for tissue engineering. Journal of Biomedical Materials Research, 2002. 60(4): p. 613-621.
5. H. Yoshimoto, Y.M. Shin, H. Terai, J.P. Vacanti, A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials, 2003. 24(12): p. 2077-2082.
6. M.S. Widmer, P.K. Gupta, L. Lu, R.K. Meszlenyi, G.R.D. Evans, K. Brandt, T. Savel, A. Gurlek, C.W. Patrick Jr, A.G. Mikos, Manufacture of porous biodegradable polymer conduits by an extrusion process for guided tissue regeneration. Biomaterials, 1998. 19(21): p. 1945-1955.
7. O.N. Ko, S.L. Gerson, Akt helps stem cells heal the heart. Nature Medicine, 2003. 9(9): p. 1109-1110.
8. M. Tomita, E. Lavik, H. Klassen, T. Zahir, R. Langer, M.J. Young, Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells, 2005. 23(10): p. 1579-1588.
9. V.J. Chen, P.X. Ma, Nano-fibrous poly(L-lactic acid) scaffolds with interconnected spherical macropores. Biomaterials, 2004. 25(11): p. 2065-2073.
10. G.A. Abrams, S.L. Goodman, P.F. Nealey, M. Franco, C.J. Murphy, Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque. Cell and Tissue Research, 2000. 299(1): p. 39-46.
11. G. Chen, T. Ushida, T. Tateishi, Hybrid biomaterials for tissue engineering: A preparative method for PLA or PLGA-collagen hybrid sponges. Advanced Materials, 2000. 12(6): p. 455-457.
12. Z. Ma, C. Gao, Y. Gong, J. Shen, Paraffin Spheres as Porogen to Fabricate Poly(L-Lactic Acid) Scaffolds with Improved Cytocompatibility for Cartilage Tissue Engineering. Journal of Biomedical Materials Research - Part B Applied Biomaterials, 2003. 67(1): p. 610-617.
13. O.N. Koc, S.L. Gerson, Akt helps stem cells heal the heart. Nature Medicine, 2003. 9(9): p. 1109-1110.
14. R.C. Hendel, T.D. Henry, K. Rocha-Singh, J.M. Isner, D.J. Kereiakes, F.J. Giordano, M. Simons, R.O. Bonow, Effect of intracoronary recombinant human vascular endothelial growth factor on myocardial perfusion: Evidence for a dose-dependent effect. Circulation, 2000. 101(2): p. 118-121.
15. T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric system for dual growth factor delivery. Nature Biotechnology, 2001. 19(11): p. 1029-1034.
16. L.D. Shea, E. Smiley, J. Bonadio, D.J. Mooney, DNA delivery from polymer matrices for tissue engineering. Nature Biotechnology, 1999. 17(6): p. 551-554.
17. K.H. Chakrabarty, M. Heaton, A.J. Dalley, R.A. Dawson, E. Freedlander, P.T. Khaw, S. Mac Neil, Keratinocyte-driven contraction of reconstructed human skin. Wound Repair Regen, 2001. 9(2): p. 95-106.
18. P.X. Ma, R. Zhang, G. Xiao, R. Franceschi, Engineering new bone tissue in vitro on highly porous poly(汐-hydroxyl acids)/hydroxyapatite composite scaffolds. Journal of Biomedical Materials Research, 2001. 54(2): p. 284-293.
19. P.X. Ma, J.W. Choi, Biodegradable polymer scaffolds with well-defined interconnected spherical pore network. Tissue Engineering, 2001. 7(1): p. 23-33.
20. P.X. Ma, R. Zhang, Synthetic nano-scale fibrous extracellular matrix. Journal of Biomedical Materials Research, 1999. 46(1): p. 60-72.
21. J.A. Burdick, M. Ward, E. Liang, M.J. Young, R. Langer, Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels. Biomaterials, 2006. 27(3): p. 452-459.
22. J.O. Winter, M. Gokhale, R.J. Jensen, S.F. Cogan, J.F. Rizzo Iii, Tissue engineering applied to the retinal prosthesis: Neurotrophin-eluting polymeric hydrogel coatings. Materials Science and Engineering C, 2008. 28(3): p. 448-453.
23. L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proceedings of the National Academy of Sciences of the United States of America, 1976. 73(7): p. 2424-2428.
24. D.E. Ingber, V.C. Mow, D. Butler, L. Niklason, J. Huard, J. Mao, I. Yannas, D. Kaplan, G. Vunjak-Novakovic, Tissue engineering and developmental biology: Going biomimetic. Tissue Engineering, 2006. 12(12): p. 3265-3283.
25. J.A. Burdick, M. Ward, E. Liang, M.J. Young, R. Langer, Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels. Biomaterials, 2006. 27(3): p. 452-459.
26. J.O. Winter, M. Gokhale, R.J. Jensen, S.F. Cogan, J.F. Rizzo Iii, Tissue engineering applied to the retinal prosthesis: Neurotrophin-eluting polymeric hydrogel coatings. Materials Science and Engineering C, 2008. 28(3): p. 448-453.
27. M. Tomita, E. Lavik, H. Klassen, T. Zahir, R. Langer, M.J. Young, Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells, 2005. 23(10): p. 1579-1588.
28. L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proceedings of the National Academy of Sciences of the United States of America, 1976. 73(7): p. 2424-2428.
29. X. Zong, K. Kim, D. Fang, S. Ran, B.S. Hsiao, B. Chu, Structure and process relationship of electrospun bioabsorbable nanofiber membranes. Polymer, 2002. 43(16): p. 4403-4412.
30. D.H. Reneker, I. Chun, Nanometre diameter fibres of polymer, produced by electrospinning. Nanotechnology, 1996. 7(3): p. 216-223.
31. W.J. Li, C.T. Laurencin, E.J. Caterson, R.S. Tuan, F.K. Ko, Electrospun nanofibrous structure: A novel scaffold for tissue engineering. Journal of Biomedical Materials Research, 2002. 60(4): p. 613-621.

------------------------------------------------------------------------ 第 6 筆 ---------------------------------------------------------------------
系統識別號 U0007-0208201011442200
論文名稱(中文) 雌激素經由調控骨橋蛋白促進肺腺癌細胞生長及移行之訊息傳遞路徑
論文名稱(英文) Estrogen Promote Tumor Cells Growth and Migration by Regulating Osteopontin Signal Pathway in Lung Adenocarcinoma
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 蔡明芳
學號 M120097006
學位類別 碩士
語文別 中文
口試日期 2010-07-02
論文頁數 85頁
口試委員 委員-劉柯俊
委員-許文瀚
指導教授-高淑慧
關鍵字(中) 雌激素
gefitinib
肺癌
MEK/ERK
骨橋蛋白
tamoxifen citrate
關鍵字(英) estrogen
gefitinib
lung cancer
MEK/ERK
osteopontin
tamoxifen citrate
學科別分類
中文摘要 在台灣,肺癌死亡率占惡性腫瘤中的首位,並有逐年增加的趨勢。由於肺癌在女性中發生率漸增且大多數女性並沒有吸菸的習慣,因此雌激素 (17β-estradiol, E2)被推測於肺癌的病理機轉具有重要的地位。另外,有研究指出,E2可以促使骨橋蛋白 (osteopontin, OPN)的表現,而OPN與腫瘤的增生、移行、侵入及轉移有關。因此,我們擬探討E2及OPN在肺癌細胞的成長及細胞移行所扮演的角色,並釐清其分子訊息路徑。此外,合併使用tamoxifen citrate及gefitinib是否能對肺癌細胞生長及移行產生更有效的抑制也是我們觀察的目標。首先,我們在A549細胞株發現E2會促使細胞生長、移行以及活化基質金屬蛋白酶 (matrix metalloproteinase, MMP-2);在PE089肺腺癌細胞株也同樣觀察到E2會引發細胞移行。而使用MEK抑制劑U0126則會抑制細胞移行顯示E2的作用是透過活化MEK/ERK訊息傳遞路徑。合併使用tamoxifen citrate及gefitinib兩種藥物處理細胞株後,發現可以更有效的抑制細胞的移行能力。在A549細胞株,E2可以促進OPN RNA的表現,而tamoxifen citrate則可以抑制此現象。此外,我們收集來自12個癌症病人肋膜積水檢體,偵測其OPN及E2的濃度。結果發現,在lung adenocarcinoma病人,肋膜積水中OPN及E2的濃度高於控制組3倍,而且OPN與E2兩者的濃度具有正相關性。以肋膜積水培養A549及PE089細胞株。結果發現,在A549細胞株,細胞移行的能力與OPN或E2並沒有正相關性;而在PE089細胞株中,細胞移行的能力與OPN有正相關性,但與E2則沒有關連。綜合實驗結果,我們推論未來臨床上合併使用tamoxifen citrate及gefitinib可以思考做為難治肺癌的一種治療方式,另外,OPN可能可以作為一個肺癌治療預測因子及預後的指標。
英文摘要 Lung cancer is the most common cause of cancer death in Taiwan. The incidence of lung cancer is increasing in female and most of them don’t smoke. Estrogen (E2) is proposed to play an important role in pathogenesis of lung cancer. Besides, previous studies reported estrogen could stimulate osteopontin (OPN) expression, and OPN has an important role in tumor proliferation, migration, invasion and metastasis. We try to explore the roles of estrogen and OPN in lung cancer cell lines growth and migration, and investigate the participated signaling pathway. The separate and combined effect of tamoxifen citrate and gefitinib on the tumor cell growth and migration was also studied. We found estrogen could stimulate A549 lung cancer cell line growth, migration and activation of MMP-2. In PE089 lung cancer cell line, estrogen could stimulate cell migration. The effect was through the activation of MEK/ERK signaling pathway. Synergistic effect of tamoxifen citrate and gefitinib on the inhibition of cell migration was noted. Estrogen could stimulate OPN RNA expression in A549 cell line, and tamoxifen citrate antagonized the effect. We collected pleural effusions from twelve cancer patients and measured their OPN and E2 concentrations. The pleural fluid OPN and E2 concentrations from patients with lung adenocarcinoma are three times more than the control. We tried adding the pleural fluid to cultured A549 and PE089 cell lines There is no correlation between pleural fluid E2 concentrations and migration in both cell lines. But, positive correlation between pleural fluid OPN concentration and migration was found in PE089 cell line. According to the above results, gefitinib combined with tamoxifen citrate may be a new treatment modality for patients with refractory lung cancer and OPN could be looked as a predictor for treatment and prognosticator.
論文目次 中文摘要 1
Abstract 3
研究動機及目標 5
第一章 文獻回顧 6
肺癌 (Lung cancer) 7
肺癌種類 7
肺癌成因 8
治療方式 10
雌激素 (Estrogen) 11
雌激素合成 11
雌激素功能 12
雌激素受體(estrogen receptor, ER) 12
雌激素受體的分子結構 12
雌激素受體的分布及功能 13
雌激素受體與肺癌的相關研究 14
骨橋蛋白 (Osteopontin) 15
第二章 實驗材料與方法 17
藥品試劑 18
實驗方法 20
一、人類肺腺癌細胞培養 20
二、檢體收集 20
三、藥物配置 21
四、細胞計數 21
五、分析細胞內RNA表現 22
六、細胞蛋白質製備(Preparation of cell lysate) 23
七、粒線體分離 (Mitochondrial fraction) 24
八、蛋白質定量法(Protein assay) 25
九、西方墨點法(Western blotting) 25
十、Scratch wound assay 27
十一、Gel zymography 27
十二、肋膜積水之骨橋蛋白(OPN)的測定 29
十三、雷射共軛顯微鏡 30
十四、統計分析 31
第三章 實驗結果與分析 32
一、 偵測肺癌細胞株中是否含有ER 33
二、 雌激素 (E2)對於肺癌細胞株生長曲線 (growth curve)、存活率(viability)的影響 33
三、 E2對於肺癌細胞株移行能力的影響 34
四、 E2對於肺癌細胞株表現MMP-2的影響 34
五、 E2藉由活化MEK-ERK訊息傳遞路徑調控細胞移行能力 35
六、 合併臨床藥物tamoxifen citrate及gefitinib對於肺癌細胞的影響 36
七、 E2對於肺癌細胞株之OPN的表現的影響 37
八、 E2透過刺激OPN表現以促進細胞移行能力 37
九、 OPN對於肺癌細胞株移行能力的影響 38
十、 偵測肋膜積水 (Pleural effusion)中OPN及E2的含量 38
十一、 肋膜積水促進細胞移行能力與OPN及E2含量的相關性 39
十二、 觀察ERβ在肺腺癌細胞中的分布情形 40
第四章 討論 41
第五章 參考文獻 51
第六章 圖表 59
第七章 附件 84
參考文獻 Atalay G, Cardoso F, Awada A, Piccart MJ (2003) Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer. Ann Oncol 14: 1346-1363

Banerjee A, Rose R, Johnson GA, Burghardt RC, Ramaiah SK (2009) The influence of estrogen on hepatobiliary osteopontin (SPP1) expression in a female rodent model of alcoholic steatohepatitis. Toxicol Pathol 37: 492-501

Brown LF, Papadopoulos-Sergiou A, Berse B, Manseau EJ, Tognazzi K, Perruzzi CA, Dvorak HF, Senger DR (1994) Osteopontin expression and distribution in human carcinomas. Am J Pathol 145: 610-623

Chang YS, Kim HJ, Chang J, Ahn CM, Kim SK (2007) Elevated circulating level of osteopontin is associated with advanced disease state of non-small cell lung cancer. Lung Cancer 57: 373-380

Cheng YW, Chiou HL, Sheu GT, Hsieh LL, Chen JT, Chen CY, Su JM, Lee H (2001) The association of human papillomavirus 16/18 infection with lung cancer among nonsmoking Taiwanese women. Cancer Res 61: 2799-2803

Craig AM, Denhardt DT (1991) The murine gene encoding secreted phosphoprotein 1 (osteopontin): promoter structure, activity, and induction in vivo by estrogen and progesterone. Gene 100: 163-171

Das R, Mahabeleshwar GH, Kundu GC (2004) Osteopontin induces AP-1-mediated secretion of urokinase-type plasminogen activator through c-Src-dependent epidermal growth factor receptor transactivation in breast cancer cells. J Biol Chem 279: 11051-11064

Denhardt DT, Noda M, O'Regan AW, Pavlin D, Berman JS (2001) Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 107: 1055-1061

Dougherty SM, Mazhawidza W, Bohn AR, Robinson KA, Mattingly KA, Blankenship KA, Huff MO, McGregor WG, Klinge CM (2006) Gender difference in the activity but not expression of estrogen receptors alpha and beta in human lung adenocarcinoma cells. Endocr Relat Cancer 13: 113-134

Egloff AM, Rothstein ME, Seethala R, Siegfried JM, Grandis JR, Stabile LP (2009) Cross-talk between estrogen receptor and epidermal growth factor receptor in head and neck squamous cell carcinoma. Clin Cancer Res 15: 6529-6540

Fedarko NS, Fohr B, Robey PG, Young MF, Fisher LW (2000) Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack. J Biol Chem 275: 16666-16672

Fedarko NS, Jain A, Karadag A, Van Eman MR, Fisher LW (2001) Elevated serum bone sialoprotein and osteopontin in colon, breast, prostate, and lung cancer. Clin Cancer Res 7: 4060-4066

Filardo EJ, Quinn JA, Bland KI, Frackelton AR, Jr. (2000) Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14: 1649-1660

Fong YC, Liu SC, Huang CY, Li TM, Hsu SF, Kao ST, Tsai FJ, Chen WC, Chen CY, Tang CH (2009) Osteopontin increases lung cancer cells migration via activation of the alphavbeta3 integrin/FAK/Akt and NF-kappaB-dependent pathway. Lung Cancer 64: 263-270

Funakoshi T, Yanai A, Shinoda K, Kawano MM, Mizukami Y (2006) G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane. Biochem Biophys Res Commun 346: 904-910

Geraldes P, Sirois MG, Bernatchez PN, Tanguay JF (2002) Estrogen regulation of endothelial and smooth muscle cell migration and proliferation: role of p38 and p42/44 mitogen-activated protein kinase. Arterioscler Thromb Vasc Biol 22: 1585-1590

Greenlee RT, Murray T, Bolden S, Wingo PA (2000) Cancer statistics, 2000. CA Cancer J Clin 50: 7-33

Hammoud Z, Tan B, Badve S, Bigsby RM (2008) Estrogen promotes tumor progression in a genetically defined mouse model of lung adenocarcinoma. Endocr Relat Cancer 15: 475-483

Havelock JC, Rainey WE, Carr BR (2004) Ovarian granulosa cell lines. Mol Cell Endocrinol 228: 67-78

Hecht SS (2003) Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer 3: 733-744

Ishijima M, Rittling SR, Yamashita T, Tsuji K, Kurosawa H, Nifuji A, Denhardt DT, Noda M (2001) Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin. J Exp Med 193: 399-404

Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, Gustafsson JA (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139: 4252-4263

Langer G, Bader B, Meoli L, Isensee J, Delbeck M, Noppinger PR, Otto C (2010) A critical review of fundamental controversies in the field of GPR30 research. Steroids 75: 603-610

Le Calvez F, Mukeria A, Hunt JD, Kelm O, Hung RJ, Taniere P, Brennan P, Boffetta P, Zaridze DG, Hainaut P (2005) TP53 and KRAS mutation load and types in lung cancers in relation to tobacco smoke: distinct patterns in never, former, and current smokers. Cancer Res 65: 5076-5083

Levin ER (2005) Integration of the extranuclear and nuclear actions of estrogen. Mol Endocrinol 19: 1951-1959

Matsuoka H, Tsubaki M, Yamazoe Y, Ogaki M, Satou T, Itoh T, Kusunoki T, Nishida S (2009) Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways. Exp Cell Res 315: 2022-2032

Matthews J, Gustafsson JA (2003) Estrogen signaling: a subtle balance between ER alpha and ER beta. Mol Interv 3: 281-292

McNamara DA, Harmey J, Wang JH, Kay E, Walsh TN, Bouchier-Hayes DJ (2001) Tamoxifen inhibits endothelial cell proliferation and attenuates VEGF-mediated angiogenesis and migration in vivo. Eur J Surg Oncol 27: 714-718
Mollerup S, Jorgensen K, Berge G, Haugen A (2002) Expression of estrogen receptors alpha and beta in human lung tissue and cell lines. Lung Cancer 37: 153-159

Niikawa H, Suzuki T, Miki Y, Suzuki S, Nagasaki S, Akahira J, Honma S, Evans DB, Hayashi S, Kondo T, Sasano H (2008) Intratumoral estrogens and estrogen receptors in human non-small cell lung carcinoma. Clin Cancer Res 14: 4417-4426

Osada H, Takahashi T (2002) Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 21: 7421-7434

Pham D, Kris MG, Riely GJ, Sarkaria IS, McDonough T, Chuai S, Venkatraman ES, Miller VA, Ladanyi M, Pao W, Wilson RK, Singh B, Rusch VW (2006) Use of cigarette-smoking history to estimate the likelihood of mutations in epidermal growth factor receptor gene exons 19 and 21 in lung adenocarcinomas. J Clin Oncol 24: 1700-1704

Prince CW, Oosawa T, Butler WT, Tomana M, Bhown AS, Bhown M, Schrohenloher RE (1987) Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J Biol Chem 262: 2900-2907

Rangaswami H, Bulbule A, Kundu GC (2004) Nuclear factor-inducing kinase plays a crucial role in osteopontin-induced MAPK/IkappaBalpha kinase-dependent nuclear factor kappaB-mediated promatrix metalloproteinase-9 activation. J Biol Chem 279: 38921-38935

Rangaswami H, Bulbule A, Kundu GC (2006) Osteopontin: role in cell signaling and cancer progression. Trends Cell Biol 16: 79-87

Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307: 1625-1630

Rittling SR, Chambers AF (2004) Role of osteopontin in tumour progression. Br J Cancer 90: 1877-1881



Sakamoto H, Matsuda K, Hosokawa K, Nishi M, Morris JF, Prossnitz ER, Kawata M (2007) Expression of G protein-coupled receptor-30, a G protein-coupled membrane estrogen receptor, in oxytocin neurons of the rat paraventricular and supraoptic nuclei. Endocrinology 148: 5842-5850

Santen RJ, Fan P, Zhang Z, Bao Y, Song RX, Yue W (2009) Estrogen signals via an extra-nuclear pathway involving IGF-1R and EGFR in tamoxifen-sensitive and -resistant breast cancer cells. Steroids 74: 586-594

Scagliotti GV, Longo M, Novello S (2009) Nonsmall cell lung cancer in never smokers. Curr Opin Oncol 21: 99-104

Schwartz AG, Prysak GM, Bock CH, Cote ML (2007) The molecular epidemiology of lung cancer. Carcinogenesis 28: 507-518

Schwartz AG, Prysak GM, Murphy V, Lonardo F, Pass H, Schwartz J, Brooks S (2005) Nuclear estrogen receptor beta in lung cancer: expression and survival differences by sex. Clin Cancer Res 11: 7280-7287

Shen H, Yuan Y, Sun J, Gao W, Shu YQ (2010) Combined tamoxifen and gefitinib in non-small cell lung cancer shows antiproliferative effects. Biomed Pharmacother 64: 88-92

Shields PG (2000) Epidemiology of tobacco carcinogenesis. Curr Oncol Rep 2: 257-262

Shigematsu H, Gazdar AF (2006) Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int J Cancer 118: 257-262

Siegfried JM (2001) Women and lung cancer: does oestrogen play a role? Lancet Oncol 2: 506-513

Siegfried JM, Hershberger PA, Stabile LP (2009) Estrogen receptor signaling in lung cancer. Semin Oncol 36: 524-531

Skov BG, Fischer BM, Pappot H (2008) Oestrogen receptor beta over expression in males with non-small cell lung cancer is associated with better survival. Lung Cancer 59: 88-94
Stabile LP, Davis AL, Gubish CT, Hopkins TM, Luketich JD, Christie N, Finkelstein S, Siegfried JM (2002) Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor alpha and beta and show biological responses to estrogen. Cancer Res 62: 2141-2150

Stabile LP, Lyker JS, Gubish CT, Zhang W, Grandis JR, Siegfried JM (2005) Combined targeting of the estrogen receptor and the epidermal growth factor receptor in non-small cell lung cancer shows enhanced antiproliferative effects. Cancer Res 65: 1459-1470

Strom A, Franzen A, Wangnerud C, Knutsson AK, Heinegard D, Hultgardh-Nilsson A (2004) Altered vascular remodeling in osteopontin-deficient atherosclerotic mice. J Vasc Res 41: 314-322

Sun J, Meyers MJ, Fink BE, Rajendran R, Katzenellenbogen JA, Katzenellenbogen BS (1999) Novel ligands that function as selective estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-beta. Endocrinology 140: 800-804

Tuck AB, Hota C, Wilson SM, Chambers AF (2003) Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways. Oncogene 22: 1198-1205

Tyan YC, Wu HY, Lai WW, Su WC, Liao PC (2005) Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry. J Proteome Res 4: 1274-1286

Wagner U, du Bois A, Pfisterer J, Huober J, Loibl S, Luck HJ, Sehouli J, Gropp M, Stahle A, Schmalfeldt B, Meier W, Jackisch C (2007) Gefitinib in combination with tamoxifen in patients with ovarian cancer refractory or resistant to platinum-taxane based therapy--a phase II trial of the AGO Ovarian Cancer Study Group (AGO-OVAR 2.6). Gynecol Oncol 105: 132-137

Wai PY, Kuo PC (2004) The role of Osteopontin in tumor metastasis. J Surg Res 121: 228-241

Wai PY, Mi Z, Guo H, Sarraf-Yazdi S, Gao C, Wei J, Marroquin CE, Clary B, Kuo PC (2005) Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis. Carcinogenesis 26: 741-751
Wang PH, Wang HC, Tsai CC (2009) Estrogen replacement in female lung cancer during gefitinib therapy. Jpn J Clin Oncol 39: 829-832

Wang YC, Lu YP, Tseng RC, Lin RK, Chang JW, Chen JT, Shih CM, Chen CY (2003) Inactivation of hMLH1 and hMSH2 by promoter methylation in primary non-small cell lung tumors and matched sputum samples. J Clin Invest 111: 887-895

White FJ, Burghardt RC, Hu J, Joyce MM, Spencer TE, Johnson GA (2006) Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation. Reproduction 132: 919-929

Wu Y, Denhardt DT, Rittling SR (2000) Osteopontin is required for full expression of the transformed phenotype by the ras oncogene. Br J Cancer 83: 156-163

Zhang G, Liu X, Farkas AM, Parwani AV, Lathrop KL, Lenzner D, Land SR, Srinivas H (2009) Estrogen receptor beta functions through nongenomic mechanisms in lung cancer cells. Mol Endocrinol 23: 146-156

Zhao Y, Wang S, Aunan K, Seip HM, Hao J (2006) Air pollution and lung cancer risks in China--a meta-analysis. Sci Total Environ 366: 500-513

Zohar R, Zhu B, Liu P, Sodek J, McCulloch CA (2004) Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway. Am J Physiol Heart Circ Physiol 287: H1730-1739

------------------------------------------------------------------------ 第 7 筆 ---------------------------------------------------------------------
系統識別號 U0007-0208201014154100
論文名稱(中文) 建立動物和細胞實驗模式以評估血腦障壁功能之研究
論文名稱(英文) ESTABLISHMENT OF ANIMAL AND CELL MODELS TO EVALUATE THE FUNCTIONS OF THE BLOOD-BRAIN BARRIER
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 謝惠姍
學號 M120097027
學位類別 碩士
語文別 中文
口試日期 2010-07-16
論文頁數 70頁
口試委員 委員-翁祖輝 教授
委員-陳大樑 教授
指導教授-陳瑞明 教授
關鍵字(中) 血腦障壁
模式
關鍵字(英) blood-brain barrier
models
學科別分類
中文摘要 血腦障壁存在週邊循環系統與中樞神經系統間,它是由一連串的大腦血管內皮細胞藉由 tight junctions所組成,負責調節離子平衡、養分運輸和阻擋有害物質進入腦中。疾病進展的過程,慢性疾病例如阿茲海默症、多發性硬化症,急性症狀例如中風或腦創傷會造成血腦障壁失去功能,進而破壞中樞神經系統。在過去的動物實驗模式中,對於疾病進展過程症狀之一慢性發炎對於血腦障壁的影響,以及急性的腦缺血再灌流後,對於血腦障壁的通透性還未有明確的研究。因此,本篇研究將建立動物及細胞實驗模式以評估血腦障壁的功能正常與否。
動物模式方面我們使用 menadione 作為活性氧物質的提供者,以模擬長時間氧化壓力傷害造成慢性發炎,雙邊總頸動脈栓塞 (Bilateral common carotid artery occlusion; BCAO) 及右側大腦中腦動脈栓塞 (middle cerebral artery occlusion ; MCAO) 的手術方式接近人發生急性缺血性中風的情況。細胞模式是將腦血管內皮細胞單獨或與星狀細胞共同培養,觀察 monolayer 完整性及 ZO-1 形成情形。結果顯示,不論menadione、 MCAO 或 BCAO 組皆會導致血腦屏障破壞,而預先給予白藜蘆醇和維他命 C 可以改善血腦屏障破壞。利用2,3,5-triphenyltetrazolium chloride (TTC) 染色觀察腦受損區域,在 MCAO 組會看到明顯白色區域,表示此區細胞壞死。此外,menadione 會造成腦血管內皮細胞及 tight junction 破壞。從細胞培養型態來看,兩種培養模式皆會形成緻密 monolayer,又以共同培養的細胞型態看起來更為緊密排列。不論是單獨培養腦血管內皮細胞或共同培養細胞在第二天開始有 ZO-1 表現,共同培養情況下會有較多的 ZO-1表現,顯示 monolayer 更為完整。
由本研究成果顯示,在動物模式上,menadione 注射模式可作為慢性發炎去影響血腦障壁的研究模式,全腦性腦損傷BCAO 模式、區域性腦損傷MCAO 模式皆會造成血腦障壁不同程度的傷害, menadione 更會藉由破壞腦內皮細胞及tight junction protein使得血腦障壁通透度上升;在細胞模式上,腦血管內皮細胞間隨天數增加 tight junction protein的形成越趨完整。綜合實驗結果,這四種模式可作為日後探討抗氧化劑對於血腦障壁保護功效及機制的模式。
英文摘要 The blood-brain barrier (BBB) is between the central nervous system (CNS) and the peripheral circulatory system mainly consists of cerebral endothelial cells (CECs) that form compact tight junctions as a selective physical barrier. It functions as a dynamic regulator of ion balance, a facilitator of nutrient transport, and a barrier to potentially harmful molecules. Disruption of the BBB is a critical event in the development and progression of several diseases that affect the CNS such as neurodegenerative disorders, stroke and traumatic brain injury. Therefore, in this study, we attempted to establish animal and cell models to evaluate the functions of the BBB.
In the animal study, we used menadione as a generator of oxidative stress. Bilateral common carotid artery occlusion (BCAO) and Right middle cerebral artery occlusion (MCAO) mimicked ischemia/reperfusion (I/R). The results suggested that menadione-treated, MCAO, BCAO groups led to BBB disruption. Pretreatment with resveratrol and vitamin C improved the BBB disruption. In addition, resveratrol enhanced numbers of zonula occludens-1 (ZO-1) compared with menadione-treated group. To provide an in vitro system for studying BBB functions, we have developed a process of culture at CECs with or without astrocytes in dishes. In order to detect tight junction formation, the cells formed monolayer and then immunofluorescent examination of ZO-1. These results showed that ZO-1 typically located in tight junctions of endothelial layers at 3nd day.
In conclusion, the BBB permeability was increased by menadione, BCAO, and MCAO. Coculture of CECs and astrocyte formed a tighter monolayer than culture of CECs did. Through characterization of the models we have established that mice are suitable for studing protective effects of the antioxidant in BBB.
論文目次 目錄………………………………………………..……….…………I
圖目次………………………………………..……….……..…….….V
表目次….……………………………………………………….........VI
摘要………………………………………..………………......……..VII
Abstract...………………………….………………….……………….IX
縮寫表..…………………………..………………………………..….XI
第一章 緒言...……..…………………...…….……….…………...1
第一節 腦疾病與血腦障壁……………………………………....1
第二節 血腦障壁與中樞神經系統………………………………3
2.1 中樞神經系統……………………………………….3
2.2 血腦障壁的組成與功能…………………………….3
2.2.1 血腦障壁組成……………………………………..3
2.2.2 Tight junction 蛋白………………………………..5
2.2.3 血腦障壁的功能…………………………………..6
第三節 腦疾病與動物實驗模式…………………………………7
第四節 研究目的與研究目標……………………………………8
4.1 研究目的…………………………………………….8
4.2 研究目標…………………………………………….8
第五節 實驗設計及說明…………………………………………9
第二章 材料與方法………………………………….……..……14
第一節 實驗材料…………………………………………………14
第二節 動物………………………………………………………14
第三節 動物實驗模式……………………………………………15
3.1 menadione 長時間誘導氧化壓力傷害……………..15
3.2雙邊頸動脈栓塞模式………………………………..15
3.3中腦動脈栓塞模式…………………………………..16
第四節 腦部傷害測定……………………………………………17
4.1小鼠腦 Evans blue 染色測量血腦障壁通透性……17
4.2 2,3,5-triphenyltetrazolium chloride 染色測量腦
梗塞範圍…………………………………………………17
第四節 免疫組織化學染色法……………………………………17
5.1 動物組織固定……………………………………….17
5.2 免疫組織化學染色法……………………………….18
第六節 腦組織蛋白表現分析……………………………………18
6.1蛋白質萃取…………………………………………..18
6.2 蛋白質定量………………………………………….19
6.3蛋白質電泳與免疫轉漬分析………………………..19
第七節 小鼠體外血腦障壁培養模式……………………….…….20
7.1小鼠腦血管內皮細胞之製備…………………………20
7.2腦血管內皮細胞單獨培養以及與神經細胞共同
培養模式…………………………………………………..21
第八節 免疫細胞化學染色法……………………………………..21
8.1 細胞固定……………………………………………...21
8.2免疫細胞化學染色法………………………..………..22
第九節 統計分析…………………………………………………..22
第三章 結果…….…….…………………….…..…………...…..…23
第一節 menadione 注射模式會造成血腦障壁通透性上升……...23
第二節 白藜蘆醇與維他命 C 保護ICR小鼠免於
menadione 注射模式所造成的血腦障壁傷害…………..23
第三節 menadione 注射模式下會減少腦血管內皮細胞和
tight junctions結構……………………………………..…24
第四節 BCAO 與 MCAO 皆會造成血腦障壁傷害…………….25
第五節 腦血管內皮細胞間 tight junctions形成………………....27
第四章 討論……………………………………………….............29
第一節 Evan blue dye 和 TTC 用以判別血腦障壁通透與
腦部受損區域…………………………………………….29
第二節 menadione 造成小鼠血腦障壁通透性上升…………....30
第三節 BCAO 及 MCAO 實驗模式增加腦障壁通透度…..….31
第四節 白梨蘆醇和維他命 C 保護血腦障壁免於
menadione 造成的傷害…………………………………31
第五節 血腦障壁通透改變相關機轉………………………..….32
第五章 參考文獻………………………………………...…….....35
第六章 圖表……………………………………..……...….……..50
參考文獻 Abbott NJ and Romero IA. Transporting therapeutics across the blood-brain barrier. Mol Med Today 1996;2:106-13.
Abbott NJ. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 2002;200:629-38.
Abbruscato TJ, Lopez SP, Roder K, Paulson JR. Regulation of blood-brain barrier Na,K,2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure. J Pharmacol Exp Ther 2004;310:459-68.
Adamson P, Etienne S, Couraud PO, Calder V, Greenwood J. Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway. J Immunol 1999;162:2964-73.
Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW. Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. The Penn State Retina Research Group. Diabetes 1998;47:1953-9.
Bazzoni G, Martinez-Estrada OM, Orsenigo F, Cordenonsi M, Citi S, Dejana E. Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin. J Biol Chem 2000;275:20520-6.
Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 1986;17:1304-8.
Begley DJ and Brightman MW. Structural and functional aspects of the blood-brain barrier. Prog Drug Res 2003;61:39-78.
Belayev L, Busto R, Zhao W, Ginsberg MD. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res 1996;739:88-96.

------------------------------------------------------------------------ 第 8 筆 ---------------------------------------------------------------------
系統識別號 U0007-0208201015560200
論文名稱(中文) 臨床檢驗報告自動驗證模式之研究-自動尿液試紙分析與顯微鏡人工判讀
論文名稱(英文) The Study of Autoverification for Automated Urinalysis with the Corresponding Manual Microscopic interpretations
校院名稱 臺北醫學大學
系所名稱(中) 醫學資訊研究所
系所名稱(英) Graduate Institute of Biomedical Informatics
學年度 98
學期 2
出版年 99
研究生(中文) 鄒佳君
學號 G158096006
學位類別 碩士
語文別 中文
口試日期 2010-06-30
論文頁數 69頁
口試委員 指導教授-劉建財
委員-邱泓文
委員-何信重
關鍵字(中) 自動驗證
半定量
尿液常規檢驗
關鍵字(英) autoverification
semi-quantitative
urinalysis
學科別分類
中文摘要 隨著臨床實驗室開始導入精實化管理(Lean),加上自動分析儀也開始朝檢驗項目整合方向發展,實驗室人員與儀器的運用不再受制於所謂專業分組,希望以更少的人員、同時使用更多台儀器,以更少時間,處理更多項目檢驗檢查與不同種類的檢體,為達到這目標,實驗室務必要將檢驗結果自動驗證流程廣泛應用於越來越多檢驗項目上。檢驗結果自動驗證的設定,其困難點不僅在於需要一定的資訊硬體系統以及一定的資訊內容,而是規則該依據哪些條件來設定?尿液常規檢查檢驗項目可以篩檢腎臟、輸尿管、膀胱或尿道的病變,是醫院中最常做的檢驗項目之一,以醫學中心來看,平均每天大約要分析300~800件以上的尿液檢體;尿液常規檢驗包括儀器分析半定量數值與人員判讀兩種檢驗結果,需要以人工逐筆執行報告覆閱後才可將報告發出;受限於現今臨床檢驗報告自動驗證所提出的方法僅限於數值型,且以生化檢驗項目為主,對於尿液常規檢驗這一類包含半定量數值的檢驗報告,目前並無較好的自動驗證流程設計可供利用。本研究利用尿液常規檢驗具相關性項目的絕對正確範圍、試劑容許偏差範圍、累計容許偏差範圍,做為報告結果值自動驗證的依據,建立臨床實驗室尿液常規檢驗項目報告自動驗證流程。運用此方式,人工覆閱報告比率為14~16%,相較於實驗室原先100%人工覆閱報告比率以及10~15%重取檢體分析比率,本研究所提出的驗證範圍設計方式更有效率,估計每天可省下花在執行尿液常規檢驗作業約100~200分鐘。
英文摘要 By implementing lean in clinical laboratories, autoverification has to be more widely applied in order to improve operational efficiency. The difficulty in setting up the autoverification process is not only the need for certain hardware and data, but also which criteria should be chosen. What are valid factors in setting up the autoverification rules? The routine urinalysis, one of the widely ordered tests, including automated analysis semi-quantitative results and microscopic reading results, all the reports need to be verified case by case manually. It is difficult to design the autoverification process of semi-quantitative reports follow the rules of numeric reports like biochemical examination.
論文目次 目錄

頁數
標題 i
審定書 ii
上網授權書 iii
國科會授權書 iv
誌謝 v
目錄 vi
表目錄 viii
圖目錄 ix
中文摘要 x
英文摘要 xii
第一章 緒論 1
1.1 前言 1
1.2 研究背景 2
1.3 研究動機 5
1.4 研究目的 7
1.5 章節結構 8
1.6 研究範圍與名詞定義 8
第二章 文獻查證 12
2.1 檢驗結果自動驗證(Autoverification) 12
2.2 尿液常規檢驗與檢驗結果自動驗證 22
第三章 研究材料與方法 30
3.1 研究架構 30
3.2 研究材料 31
3.2 研究方法 33
第四章 分析與結果 52
4.1 自動驗證試行前測 52
4.2 驗證規則正確性評估 54
4.3 自動驗證測試 56
第五章 討論 59
5.1 影響自動驗證百分比因素 59
5.2 尿液沉渣檢驗結果人員差異性 61
5.3 本研究成果 63
第六章 結論與建議 65
參考資料 66
附件 69
附件一 計算盤計算方式 69

參考文獻 [1] Duca, D.J.,”Autoverfication in a Laboratory Information System”. Laboratory Medicine, 33(1),.21-25, 2002
[2] Michel R. Langlois, Joris R. Delanghe, Sophia R. Steyaert, Karel C. Everaert, and Marc L. De Buyzere, “Automated Flow Cytometry Compared with an Automated Dipstick Reader for Urinalysis”, Clinical Chemistry 45:1, 118-122, 1999
[3] Richard S. Seaberg, “Autoverification; Implementation Schemes Instrument System, or LIS”, form: http://www.aacc.org/SiteCollectionDocuments/Divisions/lis/presentations/AACCAutoverificationPresentationRev4.pdf
[4] C. T. Yen, C. T. Liu, and T. S. Chiueh, “Study on Establishing the Criteria and Parameters for Autoverification of Numerical Clinical Laboratory Test Result”. The Journal of Taiwan Association for Medical Informatics, 17, 11-24, 2008.
[5] Autoverification of Clinical Laboratory Test Result; Approved Guideline, AUTO10-A, 2007.
[6] Joris Penders, Tom Fiers, and Joris R. Delanghe, “Quantitative evaluation of urinalysis test strip” Clinical Chemistry , 48:12, 2236-2241, 2002.
[7] S. Mayo, D. Acevedo, C. Quiñones-Torrelo, I. Canós, M. Sancho, “Clinical laboratory automated urinalysis: comparison among automated microscopy, flow cytometry, two test strips analyzers, and manual microscopic examination of the urine sediments”, Journal of Clinical Laboratory Analysis, 22:4, 262-270, 2008.
[8] Richard A. Mcpherson MD, Henry’s, “Clinical Diagnosis and Management by Laboratory Methods”, 21th edition, SAUNDERS, 2007.
[9] 曾永德,臨床鏡檢學,第二版,台北,藝軒,2004。
[10] Josep M. Queraltó Compañó, Maria Ángels Bosch Ferrer, Josep Lluís Bedini Chesa, Jaume Raventós Monjo, and Xavier Fuentes-Arderiu., ”Computers in Clinical Laboratories”, Chemistry International, 30:5, 5-8, 2008.
[11] Dar-Shong Lin, MD, Fu-Yuan Huang, MD, Nan-Chang Chiu, MD, Hsin-An Koa, MD, Han-Yang Hung, MD, Chyong-Hsin Hsu, MD, Wen-Shyang Hsieh and Dien-Ie Yang, “Counts and Standard Urinalyses for Predicting Urinary Tract Infections in Febrile Infants", Pediatr Infect Dis J, 19:3; 223-227, 2000.
[12] Narayan Torke, Leonard Boral, Tracy Nguyen, Angelo Perri, and Alan Chakrin , “Process Improvement and Operational Efficiency through Test Result Autoverification”, Clinical Chemistry 51:12, 2406-2408, 2005.
[13] Michael W. Fowler, “AUTOVERIFICATION : The HOW”, from: http://www.aacc.org/SiteCollectionDocuments/Divisions/lis/presentations/TheHow.pdf
[14] Henk M.J. Goldschmidt, “A review of autovalidation software in laboratory medicine”, Accred Qual Assur, 7:431-440, 2002.
[15] C. Ricó S, F. Cava, J. V. García-Lario, A. Hernández, N. Iglesias, C. V. Jiménez, J. Minchinela, C. Perich, “The Reference Change Value: a Proposal to Interpret Laboratory Reports in Serial Testing Based on Biological Variation”, Scand J Clin Lab Invest, 64: 175-184, 2004.
[16] Tietz, Textbook of Clinical Chemistry
[17] Multistix® 10SG Package insert, Siemens, 2005.
[18] Clinitek Atlas Operation Manual, Siemens, 2005.
[19] S. Hata, I. Ishimaru, M. Hirokari, H. Yabuutchi and S. Masuda, “Color Pattern Inspection Machine with Human Sensitivity,” Proceedings of the 10th IEEE International Workshop on Robot and Human Interactive Communication, 68-73, Sept. 2001.
[20] C. P. Liu, “Design and Development of A PCB Auto-Inspection System by Machine Vision Technology”, M.A. Thesis, Department of Industrial Engineering and Management, National Chiao Tung University, Taiwan, 2001.
[21] P. Winkel, B. E. Statland and K. Jørgensen, “Urine Microscopy, an Ill Defined Method, Examined by Multifactorial Technique”, Clinical Chemistry 20:4, 436-439, 1974.
[22] Cornelia Ottiger and Andreas R. Huber, “Quantitative Urine Particle Analysis: Integrative Approach for the Optimal Combination of Automation with UF-100 and Microscopic Review with KOVA Cell Chamber”, Clinical Chemistry 49:4, 617–623, 2003.
[23] Thomas J. Pillsworth, Jr.,Virginia M. Haver, Christine K. Abrass, and Coliene J. Delaney, “Differentiation of Renal from Non-Renal Hematuria by Microscopic Examination of Erythrocytes in Urine”, Clinical Chemistry, 33:10, 1791-1 795, 1987.
[24] Cheng Da-lin etc. ,“Comparison of 1265 urinary specimens examined by dry-chemistry method and urinary sediment examination”, Journal of Chongqing Medical University, 27:4, 2002.
[25] Tzu-I Chien, Jau-Tsuen Kao, Hui-Lan Liu, Po-Chang Lin, Jhih-Sian Hong, Han-Peng Hsieh, Miao-Ju Chien, “Urine sediment examination: A comparison of automated urinalysis systems and manual microscopy”, Clinica Chimica Acta, 384:1-2, 28-34, 2007
[26] 林宏達,血糖與尿糖,台北榮民總醫院新陳代謝科暨糖尿病人保健推廣中心,2007

------------------------------------------------------------------------ 第 9 筆 ---------------------------------------------------------------------
系統識別號 U0007-0208201016333900
論文名稱(中文) 探討 DHTS 誘導結腸直腸癌細胞凋亡之分子機轉
論文名稱(英文) Study of the molecular mechanisms of DHTS-induced apoptosis in colorectal cancer cells
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 曾方瑜
學號 M120097039
學位類別 碩士
語文別 中文
口試日期 2010-07-15
論文頁數 41頁
口試委員 指導教授-梁有志
委員-陳建和
委員-葉添順
關鍵字(中) DHTS
結腸直腸癌
細胞凋亡
ATF3
關鍵字(英) DHTS
colorectal cancer cells
apoptosis
ATF3
學科別分類
中文摘要 由中草藥萃取出來之天然物,可做為發展新抗癌藥物的來源。本篇論文所使用的藥物15,16-Dihydrotanshinone I (DHTS) 為傳統中草藥丹参之萃取物,是具有細胞毒殺活性的成分之一。根據本實驗室先前的研究發現,DHTS 透過降低 cyclin D 和 cyclin E 的表現,讓乳癌細胞停滯於 G1 期,接著再透過粒線體途徑導致細胞凋亡。另外,實驗室也證實 DHTS 可誘導細胞內質網壓力與未折疊蛋白反應造成前列腺癌細胞凋亡。在本篇論文中探討 DHTS 作用在人類結腸直腸癌細胞 SW480 和 SW620 是否具有抗癌活性,並且進一步分析其分子機制。實驗結果首先發現,SW480 及 SW620 細胞在 1 ?慊/ml DHTS 的處理之下,細胞存活率明顯的下降。利用流式細胞儀偵測 Annexin V/propidium iodide (PI)雙染的螢光強度,得知隨著 DHTS 濃度的增加而造成細胞凋亡。接著我們研究 DHTS 使結腸直腸癌細胞凋亡的分子機制,從西方墨點法的結果發現,DHTS 誘導結腸直腸癌細胞 activating transcription factor 3 (ATF3) 的表現,其為 ATF/CREB 蛋白家族成員之一。ATF3 可藉由不同刺激使其表現,並由多種訊息途徑調控之。為了闡明 DHTS 調控的訊息傳遞路徑,我們分析 mitogen-activated protein kinase (MAPK) 的活化情形,並以 MAPK 抑制劑觀察其對 ATF3 之影響。結果顯示,DHTS 活化 MAPK 路徑早於 ATF3,並且呈現持續活化的情形,而前處理 JNK 或 p38 抑制劑,可略為阻斷 DHTS 誘導 ATF3 之表現。ATF3 過量表現可使結腸直腸癌細胞 SW480 細胞凋亡顯著增加。相較之下,結腸直腸癌細胞 SW620 過度表現 ATF3,則保護細胞因 DHTS 誘導之細胞凋亡作用。直到目前為止,實驗證明 DHTS 刺激 ATF3 表現,接著引發細胞凋亡。而 ATF3 是透過 p38-, JNK- 途徑調控之。我們証實 ATF3 在癌症發展過程中具有雙重角色,由結果顯示細胞惡性度能影響 ATF3 之功能。
英文摘要 Natural products derived from Chinese medicinal plant provide a source for development of new anticancer drugs. 15,16-dihydrotanshinone I (DHTS) was found to be an active cytotoxic component from the well-known traditional Chinese medicinal plant Salvia miltiorrhiza Bunge (Tanshen). According to our previous studies have shown that DHTS downregulated cyclin D and cyclin E, resulted in G1 phase arrest in breast cancer cell lines, and then induced apoptosis through mitochondrial apoptosis pathway. Besides, another study demonstrated that DHTS could induce apoptosis of prostate cancer cells via induction of ER stress and unfold protein response. In this study, we have investigated the molecular mechanism of DHTS anti-tumor activity in human colorectal cell lines SW480 and SW620. Preliminary data exhibited that DHTS treatment 1 ?慊/ml of SW480 and SW620 cells significantly decreased cell viability. Using Annexin V/propidium iodide (PI) staining by flow cytometry, showed DHTS resulted in a concentration-dependent apoptotic cell death. We further examined the molecular mechanism of DHTS on apoptosis in colorectal adenocarcinoma, DHTS alone induced activating transcription factor 3 (ATF3) expression, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, in colorectal adenocarcinoma as determined by Western blot analysis. Expression of ATF3 is stimulated by diverse stimuli and is likely to be regulated by many signaling pathway. To elucidate the signal transduction by DHTS, the activation of mitogen-activated protein kinase (MAPK) and the effects of ATF3 expression by MAPK inhibitors were examined. We showed that DHTS-induced ATF3 was preceded by a rapid and sustained activation of MAPK. The ATF3 expression by DHTS was blocked by JNK or p38 inhibitor pretreatment. Overexpression of ATF3 resulted in significant augment of
DHTS-activated apoptosis in SW480 cells. In contrast, ectopic expression of ATF3 in SW620 cells protected the cells from DHTS-induced reduction in cell numbers. Thus far, the current study shows that DHTS stimulates ATF3 expression and subsequently induces apoptosis. These pathways are mediated through p38-, JNK-dependent pathways. We present evidence that ATF3 has a dichotomous role in cancer development, our result suggests that the degree of malignancy of the cells affects ATF3 function.
論文目次 Abstract ....................................................................................................................... III
中文摘要....................................................................................................................... V
第一章 緒論 (Introduction) ....................................................................................... 1
壹、結腸直腸癌 (Colorectal Cancer, CRC)簡介 ................................................ 1
一、結腸直腸癌之流行病學 (Epidemiology of colorectal cancer) ............ 1
二、結腸直腸癌之分類 (Classification of colorectal cancer) .................... 1
三、結腸直腸癌之分期 (Staging of colorectal cancer) .............................. 2
四、結腸直腸癌之治療 (Treatment of colorectal cancer) .......................... 4
貳、DHTS (15,16-Dihydrotanshinone I) .............................................................. 5
参、Activating transcription factor 3 (ATF3) ....................................................... 6
一、ATF/CREB 家族 (ATF/cAMP response element-binding protein family) ............................................................................................................ 6
二、ATF3之結構 (Structure of ATF3) ........................................................ 7
三、ATF3 之調控 (Regulation of ATF3) .................................................... 8
四、ATF3 之生理角色 (Physiological role of ATF3)................................. 9
肆、研究目的 (Study aim)................................................................................. 10
第二章 實驗材料與方法 (Materials and Methods) ............................................... 11
壹、細胞培養 (Cell Culture) ............................................................................. 11
貳、藥物 (Drugs) ............................................................................................... 11
参、細胞存活率分析 (MTT assay) ................................................................... 12
肆、流式細胞分析 (Flow Cytometry assay) ..................................................... 12
伍、西方墨點法 (Western Blot Analysis) ......................................................... 13
一、細胞蛋白質萃取 (Protein extraction) ................................................ 13
二、蛋白質定量分析 (Protein quantification) .......................................... 13
三、SDS-膠體電泳 (SDS-PAGE electrophoresis) .................................... 14
四、轉印 (Transfer) ................................................................................... 15
五、免疫染色 (Immunoblot) ..................................................................... 15
陸、暫時性基因轉殖 (Transient transfection) .................................................. 15
第三章 結果 (Results) .............................................................................................. 17
一、DHTS 抑制人類結腸直腸癌細胞株 (SW480、SW620) 細胞增殖之能力.............................................................................................................................. 17
二、DHTS 誘導人類結腸直腸癌細胞株 (SW480、SW620) 細胞凋亡之能力.............................................................................................................................. 17
三、DHTS 正向調控 ATF3 之表現及促使 MAPK pathways 分子磷酸化 17
四、DHTS 經由 JNK 及 p38 途徑增加 ATF3 表現 ................................... 18
五、過量表現 ATF3 調控 DHTS 誘導人類結腸直腸癌細胞凋亡作用 ...... 19
II
第四章 討論 (Discussion) ......................................................................................... 20
第五章 目次圖表 (Figure lists) ................................................................................ 25
Figure 1. Effect of DHTS on the viability of human colorectal adenocarcinoma cells. ..................................................................................................................... 25
Figure 2. Effect of DHTS on the apoptosis of human colorectal adenocarcinoma cells. ..................................................................................................................... 27
Figure 3. Dose-dependent effects of DHTS on ATF3 protein expression. .......... 28
Figure 4. Effects of DHTS on phosphorylation of MAP kinases and ATF3 protein expression in time course. ....................................................................... 30
Figure 5. The effect of MAPK inhibitors on ATF3 expression under DHTS treatment .............................................................................................................. 31
Figure 6. Effects of overexpressed ATF3 on cell death induced by DHTS. ....... 32
第六章 參考文獻 (References) ................................................................................ 34
第七章 附錄 (Appendixes) ....................................................................................... 40
參考文獻 1. McConnell EL, Liu F, Basit AW: Colonic treatments and targets: issues and opportunities. J Drug Target 2009, 17:335-363.
2. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics, 2008. CA Cancer J Clin 2008, 58:71-96.
3. Segal NH, Saltz LB: Evolving treatment of advanced colon cancer. Annu Rev Med 2009, 60:207-219.
4. Compton CC: Colorectal carcinoma: diagnostic, prognostic, and molecular features. Mod Pathol 2003, 16:376-388.
5. Royston D, Jackson DG: Mechanisms of lymphatic metastasis in human colorectal adenocarcinoma. J Pathol 2009, 217:608-619.
6. Mainprize KS, Mortensen NJ, Warren BF: Early colorectal cancer: recognition, classification and treatment. Br J Surg 1998, 85:469-476.
7. Haubrich WS: Dukes of the Dukes classification. Gastroenterology 2000, 118:828.
8. Leibovitz A, Stinson JC, McCombs WB, 3rd, McCoy CE, Mazur KC, Mabry ND: Classification of human colorectal adenocarcinoma cell lines. Cancer Res 1976, 36:4562-4569.
9. Zlobec I, Lugli A: Prognostic and predictive factors in colorectal cancer. J Clin Pathol 2008, 61:561-569.
10. Weitz J, Koch M, Debus J, Hohler T, Galle PR, Buchler MW: Colorectal cancer. Lancet 2005, 365:153-165.
11. Compton CC: Colorectal Carcinoma: Diagnostic, Prognostic, and Molecular Features. Mod Pathol, 16:376-388.
12. Wang X, Morris-Natschke SL, Lee KH: New developments in the chemistry and biology of the bioactive constituents of Tanshen. Med Res Rev 2007, 27:133-148.
13. Tzen JT, Jinn TR, Chen YC, Li FY, Cheng FC, Shi LS, She H, Chen BC, Hsieh V, Tu ML: Magnesium lithospermate B possesses inhibitory activity on Na+,K+-ATPase and neuroprotective effects against ischemic stroke. Acta Pharmacol Sin 2007, 28:609-615.
14. Han J-Y, Fan J-Y, Horie Y, Miura S, Cui D-H, Ishii H, Hibi T, Tsuneki H, Kimura I: Ameliorating effects of compounds derived from Salvia miltiorrhiza root
35
extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion. Pharmacology & Therapeutics 2008, 117:280-295.
15. Tsai SL, Suk FM, Wang CI, Liu DZ, Hou WC, Lin PJ, Hung LF, Liang YC: Anti-tumor potential of 15,16-dihydrotanshinone I against breast adenocarcinoma through inducing G1 arrest and apoptosis. Biochem Pharmacol 2007, 74:1575-1586.
16. Jiang RW, Lau KM, Hon PM, Mak TC, Woo KS, Fung KP: Chemistry and biological activities of caffeic acid derivatives from Salvia miltiorrhiza. Curr Med Chem 2005, 12:237-246.
17. Lee P, Hur J, Lee J, Kim J, Jeong J, Kang I, Kim SY, Kim H: 15,16-dihydrotanshinone I suppresses the activation of BV-2 cell, a murine microglia cell line, by lipopolysaccharide. Neurochem Int 2006, 48:60-66.
18. Jeon SJ, Son KH, Kim YS, Choi YH, Kim HP: Inhibition of prostaglandin and nitric oxide production in lipopolysaccharide-treated RAW 264.7 cells by tanshinones from the roots of Salvia miltiorrhiza bunge. Arch Pharm Res 2008, 31:758-763.
19. Lee WY, Liu KW, Yeung JH: Reactive oxygen species-mediated kinase activation by dihydrotanshinone in tanshinones-induced apoptosis in HepG2 cells. Cancer Lett 2009, 285:46-57.
20. Allan AL, Albanese C, Pestell RG, LaMarre J: Activating transcription factor 3 induces DNA synthesis and expression of cyclin D1 in hepatocytes. J Biol Chem 2001, 276:27272-27280.
21. Hai T, Curran T: Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A 1991, 88:3720-3724.
22. Hill CS, Treisman R: Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 1995, 80:199-211.
23. Lee KA, Hai TY, SivaRaman L, Thimmappaya B, Hurst HC, Jones NC, Green MR: A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters. Proc Natl Acad Sci U S A 1987, 84:8355-8359.
24. Montminy M: Transcriptional regulation by cyclic AMP. Annu Rev Biochem 1997, 66:807-822.
25. Lin YS, Green MR: Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters. Proc Natl Acad Sci U S A 1988, 85:3396-3400.
26. Hai T, Hartman MG: The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of
36
transcription factors: activating transcription factor proteins and homeostasis. Gene 2001, 273:1-11.
27. Hai TW, Liu F, Coukos WJ, Green MR: Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev 1989, 3:2083-2090.
28. Chinenov Y, Kerppola TK: Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 2001, 20:2438-2452.
29. Hai T, Wolfgang CD, Marsee DK, Allen AE, Sivaprasad U: ATF3 and stress responses. Gene Expr 1999, 7:321-335.
30. Liang G, Wolfgang CD, Chen BP, Chen TH, Hai T: ATF3 gene. Genomic organization, promoter, and regulation. J Biol Chem 1996, 271:1695-1701.
31. Hsu JC, Laz T, Mohn KL, Taub R: Identification of LRF-1, a leucine-zipper protein that is rapidly and highly induced in regenerating liver. Proc Natl Acad Sci U S A 1991, 88:3511-3515.
32. Shtil AA, Mandlekar S, Yu R, Walter RJ, Hagen K, Tan TH, Roninson IB, Kong AN: Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells. Oncogene 1999, 18:377-384.
33. Zimmermann J, Erdmann D, Lalande I, Grossenbacher R, Noorani M, Furst P: Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1. Oncogene 2000, 19:2913-2920.
34. Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S, Ichijo H, Kitajima S: Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH(2)-terminal kinase and promoter response element. Blood 2000, 96:2140-2148.
35. Nawa T, Nawa MT, Cai Y, Zhang C, Uchimura I, Narumi S, Numano F, Kitajima S: Repression of TNF-alpha-induced E-selectin expression by PPAR activators: involvement of transcriptional repressor LRF-1/ATF3. Biochem Biophys Res Commun 2000, 275:406-411.
36. Turchi L, Fareh M, Aberdam E, Kitajima S, Simpson F, Wicking C, Aberdam D, Virolle T: ATF3 and p15PAF are novel gatekeepers of genomic integrity upon UV stress. Cell Death Differ 2009, 16:728-737.
37. Kool J, Hamdi M, Cornelissen-Steijger P, van der Eb AJ, Terleth C, van Dam H: Induction of ATF3 by ionizing radiation is mediated via a signaling pathway that includes ATM, Nibrin1, stress-induced MAPkinases and ATF-2. Oncogene 2003, 22:4235-4242.
37
38. Hartman MG, Lu D, Kim ML, Kociba GJ, Shukri T, Buteau J, Wang X, Frankel WL, Guttridge D, Prentki M, et al: Role for activating transcription factor 3 in stress-induced beta-cell apoptosis. Mol Cell Biol 2004, 24:5721-5732.
39. Francis JS, Dragunow M, During MJ: Over expression of ATF-3 protects rat hippocampal neurons from in vivo injection of kainic acid. Brain Res Mol Brain Res 2004, 124:199-203.
40. Lu D, Wolfgang CD, Hai T: Activating transcription factor 3, a stress-inducible gene, suppresses Ras-stimulated tumorigenesis. J Biol Chem 2006, 281:10473-10481.
41. Kashiwakura Y, Ochiai K, Watanabe M, Abarzua F, Sakaguchi M, Takaoka M, Tanimoto R, Nasu Y, Huh NH, Kumon H: Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and Smad regulates REIC/Dickkopf-3-induced apoptosis. Cancer Res 2008, 68:8333-8341.
42. Nobori K, Ito H, Tamamori-Adachi M, Adachi S, Ono Y, Kawauchi J, Kitajima S, Marumo F, Isobe M: ATF3 inhibits doxorubicin-induced apoptosis in cardiac myocytes: a novel cardioprotective role of ATF3. J Mol Cell Cardiol 2002, 34:1387-1397.
43. Su CC, Chen GW, Lin JG: Growth inhibition and apoptosis induction by tanshinone I in human colon cancer Colo 205 cells. Int J Mol Med 2008, 22:613-618.
44. Yang SY, Sales KM, Fuller B, Seifalian AM, Winslet MC: Apoptosis and colorectal cancer: implications for therapy. Trends Mol Med 2009, 15:225-233.
45. Lin A, Karin M: NF-kappaB in cancer: a marked target. Semin Cancer Biol 2003, 13:107-114.
46. Baldwin AS: Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest 2001, 107:241-246.
47. Luo JL, Kamata H, Karin M: IKK/NF-kappaB signaling: balancing life and death--a new approach to cancer therapy. J Clin Invest 2005, 115:2625-2632.
48. Karin M, Cao Y, Greten FR, Li ZW: NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002, 2:301-310.
49. Shishodia S, Aggarwal BB: Nuclear factor-kappaB: a friend or a foe in cancer? Biochem Pharmacol 2004, 68:1071-1080.
50. Lind DS, Hochwald SN, Malaty J, Rekkas S, Hebig P, Mishra G, Moldawer LL, Copeland EM, 3rd, Mackay S: Nuclear factor-kappa B is upregulated in colorectal cancer. Surgery 2001, 130:363-369.
51. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN: Modulation of
38
apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998, 58:362-366.
52. Grosch S, Maier TJ, Schiffmann S, Geisslinger G: Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 2006, 98:736-747.
53. Sinicrope FA: Targeting cyclooxygenase-2 for prevention and therapy of colorectal cancer. Mol Carcinog 2006, 45:447-454.
54. Bottone FG, Jr., Martinez JM, Collins JB, Afshari CA, Eling TE: Gene modulation by the cyclooxygenase inhibitor, sulindac sulfide, in human colorectal carcinoma cells: possible link to apoptosis. J Biol Chem 2003, 278:25790-25801.
55. Bottone FG, Jr., Moon Y, Kim JS, Alston-Mills B, Ishibashi M, Eling TE: The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3). Mol Cancer Ther 2005, 4:693-703.
56. Yan C, Jamaluddin MS, Aggarwal B, Myers J, Boyd DD: Gene expression profiling identifies activating transcription factor 3 as a novel contributor to the proapoptotic effect of curcumin. Mol Cancer Ther 2005, 4:233-241.
57. Syed V, Mukherjee K, Lyons-Weiler J, Lau KM, Mashima T, Tsuruo T, Ho SM: Identification of ATF-3, caveolin-1, DLC-1, and NM23-H2 as putative antitumorigenic, progesterone-regulated genes for ovarian cancer cells by gene profiling. Oncogene 2005, 24:1774-1787.
58. Haagenson KK, Wu GS: The role of MAP kinases and MAP kinase phosphatase-1 in resistance to breast cancer treatment. Cancer Metastasis Rev, 29:143-149.
59. Junttila MR, Li SP, Westermarck J: Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival. FASEB J 2008, 22:954-965.
60. Yang H, Choi HJ, Park SH, Kim JS, Moon Y: Macrophage inhibitory cytokine-1 (MIC-1) and subsequent urokinase-type plasminogen activator mediate cell death responses by ribotoxic anisomycin in HCT-116 colon cancer cells. Biochem Pharmacol 2009, 78:1205-1213.
61. Inoue K, Zama T, Kamimoto T, Aoki R, Ikeda Y, Kimura H, Hagiwara M: TNFalpha-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells. Genes Cells 2004, 9:59-70.
62. Mashima T, Udagawa S, Tsuruo T: Involvement of transcriptional repressor ATF3 in acceleration of caspase protease activation during DNA damaging agent-induced apoptosis. J Cell Physiol 2001, 188:352-358.
39
63. Nawa T, Nawa MT, Adachi MT, Uchimura I, Shimokawa R, Fujisawa K, Tanaka A, Numano F, Kitajima S: Expression of transcriptional repressor ATF3/LRF1 in human atherosclerosis: colocalization and possible involvement in cell death of vascular endothelial cells. Atherosclerosis 2002, 161:281-291.
64. Nakagomi S, Suzuki Y, Namikawa K, Kiryu-Seo S, Kiyama H: Expression of the activating transcription factor 3 prevents c-Jun N-terminal kinase-induced neuronal death by promoting heat shock protein 27 expression and Akt activation. J Neurosci 2003, 23:5187-5196.
65. Yin X, Dewille JW, Hai T: A potential dichotomous role of ATF3, an adaptive-response gene, in cancer development. Oncogene 2008, 27:2118-2127.
66. Mungrue IN, Pagnon J, Kohannim O, Gargalovic PS, Lusis AJ: CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade. J Immunol 2009, 182:466-476.
67. Kwok S, Rittling SR, Partridge NC, Benson CS, Thiyagaraj M, Srinivasan N, Selvamurugan N: Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells. J Cell Biochem 2009, 108:408-414.
68. Turchi L, Aberdam E, Mazure N, Pouyssegur J, Deckert M, Kitajima S, Aberdam D, Virolle T: Hif-2alpha mediates UV-induced apoptosis through a novel ATF3-dependent death pathway. Cell Death Differ 2008, 15:1472-1480.


------------------------------------------------------------------------ 第 10 筆 ---------------------------------------------------------------------
系統識別號 U0007-0208201019560200
論文名稱(中文) 製備及分析可辨認卵巢癌相關抗原的單鏈變異片段抗體
論文名稱(英文) Preparation and analysis of the single chain variable fragment antibodies recognizing the ovarian tumor associated antigen
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 林佩璇
學號 M120097051
學位類別 碩士
語文別 中文
口試日期 2010-06-28
論文頁數 70頁
口試委員 指導教授-楊沂淵
共同指導教授-呂思潔
委員-劉柯俊
委員-施能耀
關鍵字(中) 卵巢癌
關鍵字(英) ovarian tumor
學科別分類
中文摘要  卵巢癌是女性十大癌症之ㄧ,目前臨床大都以「CA-125」作為卵巢癌表現的腫瘤標記,但是靈敏度並不令人滿意。根據先前的研究利用卵巢癌病人血清中的抗體從peptide library進行篩選顯示OVTA-1可能是卵巢癌的腫瘤相關蛋白基因。本篇研究目的希望藉由噬菌體展現技術(phage display technique)製作並篩選出具有OVTA-1特異性結合反應的多株抗體與單株抗體。根據Gene Bank所公布OVTA-1的基因序列全長為3792 base pairs (bps),可轉譯出1264個胺基酸。本實驗重組其中長度為1002bp片段DNA,選殖入pET-21a質體,再利用大腸桿菌表現出與His融合的OVTA-1蛋白。西方墨點法實驗結果顯示抗His的抗體可辨識到His-OVTA-1融合蛋白。純化後的His-OVTA-1融合蛋白,以皮下注射方式注射來亨雞大腿部位,由雞蛋中純化出免疫球蛋白(IgY),再利用酵素連結免疫吸附分析法(Enzyme-Linked Immunosorbent Assay, ELISA)以及西方墨點法證實免疫過後雞隻所誘發出來的抗體可辨認His-OVTA-1片段蛋白。為了建構出可與His-OVTA-1蛋白結合的scFv噬菌體基因庫,首先自雞脾臟中萃取出total RNA反轉錄成cDNA後利用聚合酶鏈鎖反應(PCR),做出雞隻抗體的重鏈和輕鏈變異區基因片段,最後將這些基因片段連結後接入pComb3x載體中。未來希望能利用此基因庫作為OVTA-1研究和檢驗治療用之抗體來源,並探討其應用於預防及治療之潛力。
英文摘要 Ovarian cancer is one in top ten female killer cancers. CA-125 protein is generally used as a clinical tumor label of ovarian cancer; however the sensitivity is not satisfied. Previous researches showed OVTA-1 might be an ovarian cancer related gene by screening patients’ serum with the peptide library. This research aimed to construct a polyclonal or monoclonal antibody which specifically binds to OVTA-1 by phage display technique. According to the gene bank of NCBI, the full length of OVTA-1 gene is 3792bp being translated into 1264 amino acids. We cloned partial OVTA-1 gene in the length of 1002bp into pET-21a vector. The OVTA-1 protein fused with His protein would be performed by E.coli then. Results of western blot showed anti-His antibody would recognize His-OVTA-1 fusion protein. Purified His-OVTA-1 fusion protein was subcutaneous injected into leghorn chicken thighs, and immunoglobin Y (IgY) would be purified from their eggs. Next, ELISA and western blot were used to demonstrate antibodies from immunized chickens could also recognize His-OVTA-1 fusion protein. To construct anti-His-OVTA-1 scFv phagemid gene bank, total RNA was first extracted from chicken’s spleen and reverse transcribed into cDNA. Next the gene fragments of chicken antibodies’ heavy chain and light chain variable regions were amplified with PCR and ligated. At last, these ligated genes were cloned into pComb3x vector and expressed. In the future, we expect this gene bank to be the antibody source for OVTA-1 researches, clinical examinations or treatments by studying its application and therapeutic potential.
論文目次 目錄………………………………………………………………………i
圖表目錄…………………………………………………………………v
中文摘要…………………………………………………………………I
英文摘要……………………………………………………………… II
壹. 序論………………………………………………………………… 1
一. 卵巢癌與相關抗原OVTA-1簡介………………………………1
二. 以雞隻為動物模式…………………………………………… 1
三. 蛋黃免疫球蛋白(immunoglobulin Y; IgY) 簡介………… 2
四. 單株抗體簡介………………………………………………… 2
五. 利用噬菌體展現技術於抗體基因組庫選殖單株抗體的應用 …………………………………………………………… 4
貳. 實驗目的…………………………………………………………… 6
參. 實驗材料與方法…………………………………………………… 7
一. 實驗材料……………………………………………………… 7
1. 化學藥品……………………………………………………… 7
2. 酵素類………………………………………………………… 8
3. 抗體類………………………………………………………… 8
4. 其他…………………………………………………………… 9
5. 各式溶液……………………………………………………… 9
二. 實驗方法………………………………………………………… 12
1. 設計引子(Primer Design) …………………………………12
2. 選殖OVTA-1基因片段………………………………………12
3. His-OVTA-1蛋白表現…………………………………… 12
4. His-OVTA-1蛋白純化…………………………………… 13
5. 免疫注射雞隻…………………………………………………14
6. 純化蛋黃內免疫球蛋白 (IgY) ………………………………14
7. 經免疫產生的IgY 與表現的 OVTA-1-1002 蛋白片段的結合反應……………………………………………………………15
8. 建構雞隻cDNA資料庫………………………………………15
9. 抗OVTA-1-1002蛋白之scFv的基因庫建立……………… 16
10. 抗OVTA-1-1002蛋白之scFv的基因庫的篩選…………… 18
11. 表現篩選後OVTA-1-1002蛋白抗體scFv片段…………… 19
12. OVTA-1-1002蛋白抗體scFv的片段序列分析…………… 19
13.西方墨點法分析抗體片段與OVTA-1-1002
蛋白抗原之結合力…………………………………………… 19
14. 以酵素結合免疫分析法(ELISA)測試scFv抗體片段與OVTA-1-1002蛋白結合效果…………………………………19
15. 純化OVTA-1-1002重組scFv抗體片段…………………… 20
肆. 實驗結果 ………………………………………………………… 21
一. 選殖OVTA-1基因片段……………………………………… 21
二. 表現OVTA-1基因片段的蛋白……………………………… 21
三. 純化蛋黃內免疫球蛋白(IgY)……………………………… 21
四. 利用西方墨點法分析並偵測雞蛋中抗體(poly-IgY)與
OVTA-1-1002抗 原結合特異性的反應…………………… 22
五. 利用ELISA偵測抗體IgY與OVTA-1-1002抗原結合特異性的 反應………………………………………………………… 22
六. 分離脾臟並抽取RNA……………………………………… 22
七.抗OVTA-1-1002抗原之scFv基因庫的建立……………… 22
八. 確認重組抗體DNA送入XL-1 Blue E.coli中………… 23
九. 評估以噬菌體展現技術篩選到OVTA-1-1002重組蛋白抗體
基因庫片段之效價………………………………………… 23
十. 評估以噬菌體展現技術所篩選出的重組蛋白抗體基因庫片
段(scFv)與OVTA-1-1002蛋白特異性結合力之ㄧ致性… 23
十一. 表現經過篩選的OVTA-1-1002 蛋白抗體scFv片段……23
十二. 序列分析OVTA-1-1002蛋白scFv抗體片段…………… 23
十三. 利用西方墨點法分析OVTA-1-1002重組scFv抗體片段與
抗原 OVTA-1-1002蛋白結合反應………………………24
十四. 利用酵素連結免疫反應(ELISA)分析OVTA-1-1002重組
scFv抗體片段與抗原OVTA-1-1002蛋白結合反應…… 24
十五. 純化OVTA-1-1002重組scFv抗體片段………………… 24
伍. 討論 ……………………………………………………………… 25
一. OVTA-1 primer設計與OVTA-1的蛋白表現……………… 25
二. 免疫注射雞隻與萃取雞蛋中的IgY(poly-IgY)……………25
三. 抗體IgY與OVTA-1蛋白間的結合反應 ……………………26
四. 建構scFv重組抗體基因庫………………………………… 26
五. 純化scFv抗體蛋白………………………………………… 27
六. 抗體序列分析 ……………………………………………… 27
七. scFv抗體與抗原OVTA-1-1002蛋白間的結合反應……… 28
陸. 未來展望 ……………………………………………………… 29
柒. 圖表 ………………………………………………………………30
捌. 附錄……………………………………………………………… 54
玖. 參考文獻 ………………………………………………………… 67






圖 表 目 錄
Figure 1. The hydropathy profile of OVTA-1 gene……………………………….30
Figure 2. The cloning strategy for various OVTA-1 gene fragments……………31
Figure 3. The PCR products of truncated OVTA-1 gene fragments…………….32
Figure 4. Restriction analysis of recombinant DNAs containing various OVTA-1 gene fragments…………………………………………………………...33
Figure 5. Purification and characterization of truncated OVTA-1 fragments…34
Figure 6. Purification and characterization of total IgY antibodies in the eggs of chicken immunized with OVTA-1-1002 fragment……………………..35
Figure 7. Western blot analysis of purified anti-OVTA-1-1002 IgY antibodies...36
Figure 8. ELISA analysis of purified IgY antibodies on the recognition of OVTA-1-1002 protein……………………………………………………37
Figure 9. The morphology of spleen removed from a chicken immunized with His-OVTA-1-1002 protein……………………………………………….38
Figure 10. PCR products of immunoglobulin heavy and light chain variable region genes in chicken……………………………..…………………..39
Figure 11. DNA analysis on 15 randomly selected clones from original scFv-long linker library……..……………………………………………………..40
Figure 12. DNA isolation and analysis of randomly selected clones……………..41
Figure 13. Identification of scFv antibody expression……………………………42
Figure 14. comparison of the amino acid sequences of the light and heavy chain genes of scFv antibodies………………………………………………..44
Figure15. Binding ability of recombinant scFv antibodies to OVTA-1-1002 protein…………………………..……………………………………….45
Figure 16.Binding activity of antibody fragment (scFv) determined by ELISA..46
Figure 17. Purification of scFv antibody fragments protein with Ni2+ beads…...47
Table 1. Primers and their nucleotide sequences used in the amplification of different fragments of OVTA-1 gene……………………………………..49
Table 2. Schedule for immunizing chickens……………………………………….50
Table 3. Primer sequence used in the amplification VH and VL………………….51
Table 4. Primer sequence used in overlap extension PCR……………………….51
Table 5. Titers of phages expressing anti-OVTA-1-1002 scFv long linker after each panning……………………………………………………………….52
Table 6. comparison of the amino acid sequences of the light and heavy chain genes of scFv antibodies…………………………………………………53
Supplement 1. The gene map of pET-21a(+)………………………………………….55
Supplement 2. The full length of OVTA-1 gene is 3792 bp………………………56
Supplement 3. Using different IgY antibodies binding to different OVTA-1-fragment antigens………………………………………57
Supplement 4. DNA analysis on 15 randomly selected colonies from original OVTA-1-1002 scFv-short linker library………………………..59
Supplement 5. DNA isolation of randomly selected colonies…………………….60
Supplement 6. Phage Titers of OVTA-1-1002 scFv short linker after each panning…………………………………………………………….61
Supplement 7. ELISA analysis of IgY antibodies on the recognization of OVTA-1-1200 protein……………………………………………62
Supplement 8 . Purification and characterization of total IgY antibodies in the eggs of chicken immunized with OVTA-1-951 fragment……….63
Supplement 9 .Antigen binding activity of anti-OVTA-1-951 IgY antibodies purified from chicken egg yolk…………………………………...64
Supplement 10. Purification and characterization of total IgY antibodies in the eggs of chicken immunized with OVTA-1-648 fragment……..65
參考文獻 1. Quirk, J.T. and N. Natarajan, Ovarian cancer incidence in the United States, 1992-1999. Gynecologic Oncology, 2005. 97(2): p. 519-523.
2. 林明泉, 臨床血清免疫學. 2 ed, 台北市: 藝軒圖書出版社.
3. Jemal, A., et al., Cancer Statistics, 2002. CA Cancer J Clin, 2002. 52(1): p. 23-47.
4. Tuxen, M.K., et al., Assessment of Biological Variation and Analytical Imprecision of CA 125, CEA, and TPA in Relation to Monitoring of Ovarian Cancer. Gynecologic Oncology, 1999. 74(1): p. 12-22.
5. Chen, C.L., J.E. Lehmeyer, and M.D. Cooper, Evidence for an IgD homologue on chicken lymphocytes. J Immunol, 1982. 129(6): p. 2580-5.
6. Burns, R. and M. Maxwell, Probable occurrence of IgE in the adult domestic fowl (Gallus domesticus) after horse serum stimulation. Veterinary Research Communications, 1981. 5(1): p. 67-72.
7. Leslie, G.A. and L.W. Clem, PHYLOGENY OF IMMUNOGLOBULIN STRUCTURE AND FUNCTION: III. IMMUNOGLOBULINS OF THE CHICKEN. The Journal of Experimental Medicine, 1969. 130(6): p. 1337-1352.
8. Larsson A, B.R., Lindahl TL, Forsberg PO., Chicken antibodies: taking advantage of evolution--a review. Poultry Science 1993. 72(10): p. 7.
9. Donermeyer, J.W.W.a.D.D., The Interaction of Bovine Serum Albumin and Its Chicken Antibodies J. Biol. Chem., 1962. 237: p. 8.
10. Gassmann, M., et al., Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J., 1990. 4(8): p. 2528-2532.
11. Hansen, P., et al., Isolation and purification of immunoglobulins from chicken eggs using thiophilic interaction chromatography. Journal of Immunological Methods, 1998. 215(1-2): p. 1-7.
12. Schade, R., W. Burger, T. Schoneberg, A. Schniering, C. Schwarzkopf, A. Hlinak, and H. Kobilke, Avian egg yolk antibodies. The egg laying capacity of hens following immunisation with antigens of different kind and origin and the efficiency of egg yolk antibodies in comparison to mammalian antibodies. Altex, 1994. 11: p. 10.
13. Latreille, J., et al., Dexamethasone improves the efficacy of granisetron in the first 24 h following high-dose cisplatin chemotherapy. Supportive Care in Cancer, 1995. 3(5): p. 307-312.
14. Carlander, D., et al., Peroral immunotheraphy with yolk antibodies for the prevention and treatment of enteric infections. Immunologic Research, 2000. 21(1): p. 1-6.
15. valos-Pantoja, L., et al., A comparative study between the adsorption of IgY and IgG on latex particles. Journal of Biomaterials Science, Polymer Edition, 2000. 11: p. 657-673.
16. Hoffman, N., et al., Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries. Molecular Diversity, 1996. 2(1): p. 5-12.
17. D. Hadge, D. and H. Ambrosius, Evolution of low molecular weight immunoglobulins--IV. IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Molecular Immunology, 1984. 21(8): p. 699-707.
18. Sarker, S.A., et al., Randomized, Placebo-Controlled, Clinical Trial of Hyperimmunized Chicken Egg Yolk Immunoglobulin in Children With Rotavirus Diarrhea. Journal of Pediatric Gastroenterology and Nutrition, 2001. 32(1): p. 19-25.
19. Sunwoo HH, N.T., Dixon WT, Sim JS., Immune responses in chickens against lipopolysaccharide of Escherichia coli and Salmonella typhimurium. Poult Sci. , 1996 Poult Sci. (3): p. 3.
20. Fryer, J., et al., IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding. Xenotransplantation, 1999. 6(2): p. 98-109.
21. Erhard, M., et al., Adjuvant effects of various lipopeptides and interferon-gamma on the humoral immune response of chickens. Poult Sci, 2000. 79(9): p. 1264-1270.
22. Woof, J.M. and D.R. Burton, Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol, 2004. 4(2): p. 89-99.
23. Schwaber, J. and E.P. Cohen, Human [times] Mouse Somatic Cell Hybrid Clone secreting Immunoglobulins of both Parental Types. Nature, 1973. 244(5416): p. 444-447.
24. Kohler, G. and C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975. 256(5517): p. 495-497.
25. Rader, C., Antibody libraries in drug and target discovery. Drug Discovery Today, 2001. 6(1): p. 36-43.
26. Winter, G. and C. Milstein, Man-made antibodies. Nature, 1991. 349(6307): p. 293-299.
27. Kipps, T.J., et al., Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies. The Journal of Experimental Medicine, 1985. 161(1): p. 1-17.
28. Jaenisch, R. and B. Mintz, Simian Virus 40 DNA Sequences in DNA of Healthy Adult Mice Derived from Preimplantation Blastocysts Injected with Viral DNA. Proceedings of the National Academy of Sciences of the United States of America, 1974. 71(4): p. 1250-1254.
29. Kieke, M., et al., Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng., 1997. 10(11): p. 1303-1310.
30. Daugherty, P., et al., Antibody affinity maturation using bacterial surface display. Protein Eng., 1998. 11(9): p. 825-832.
31. Hanes, J., et al., Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95(24): p. 14130-14135.
32. Frykberg, K.J.a.L., Cloning of Ligand-Binding Domains of Bacterial Receptors by Phage Display Biotechniques, 1995. 18(5): p. 878-85.
33. Holliger, P. and P.J. Hudson, Engineered antibody fragments and the rise of single domains. Nat Biotech, 2005. 23(9): p. 1126-1136.
34. Smith, G., Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985. 228(4705): p. 1315-1317.
35. Barbas, C.F., et al., Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(18): p. 7978-7982.
36. Azzazy, H.M.E. and W.E. Highsmith, Phage display technology: clinical applications and recent innovations. Clinical Biochemistry, 2002. 35(6): p. 425-445.
37. Clackson, T., et al., Making antibody fragments using phage display libraries. Nature, 1991. 352(6336): p. 624-628.
38. McCafferty, J., et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 1990. 348(6301): p. 552-554.
39. A D Griffiths, M.M., J D Marks, J M Bye, M J Embleton, J McCafferty, M Baier, K P Holliger, B D Gorick, N C Hughes-Jones, Human anti-self antibodies with high specificity from phage display libraries. EMBO J, 1993. 12(2): p. 725-734.
40. Fernandez-Gacio, A., M. Uguen, and J. Fastrez, Phage display as a tool for the directed evolution of enzymes. Trends in Biotechnology, 2003. 21(9): p. 408-414.
41. Scott, J. and G. Smith, Searching for peptide ligands with an epitope library. Science, 1990. 249(4967): p. 386-390.
42. Rondot, S., et al., A helper phage to improve single-chain antibody presentation in phage display. Nat Biotech, 2001. 19(1): p. 75-78.
43. Carlos F Barbass, I., Dennis R. Burton,Jamie K. Scott,Gregg J. Silverman, Phage Display: A Laboratory Manual 2001.
44. AKITA, E.M. and S. NAKAI, Immunoglobulins from Egg Yolk: Isolation and Purification. Journal of Food Science, 1992. 57(3): p. 629-634.
45. Manoutcharian, K., et al., Phage-Displayed T-Cell Epitope Grafted into Immunoglobulin Heavy-Chain Complementarity-Determining Regions: an Effective Vaccine Design Tested in Murine Cysticercosis. Infect. Immun., 1999. 67(9): p. 4764-4770.

------------------------------------------------------------------------ 第 11 筆 ---------------------------------------------------------------------
系統識別號 U0007-0307201014300600
論文名稱(中文) 人體內臟幼蟲移行症之病原犬蛔蟲誘發Jurkat T lymphocytes進行凋亡之分子機制
論文名稱(英文) Molecular mechanisms of human visceral larva migrans pathogen Toxocara canis inducing apoptosis in Jurkat T lymphocytes.
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 周嘉玫
學號 M120097059
學位類別 碩士
語文別 中文
口試日期 2010-06-25
論文頁數 54頁
口試委員 指導教授-范家堃
委員-蕭信宏
委員-葉光勝
關鍵字(中) 犬蛔蟲
排泌
性抗原
細胞凋亡
Human Jurkat T lymphocyte
關鍵字(英) Toxocara canis
TcES
apoptosis
Human Jurkat T lymphocyte
學科別分類
中文摘要 犬蛔蟲是人畜共通寄生蟲病常見的感染原之一,人類可因誤食犬蛔蟲受孕蟲卵而感染而造成犬蛔蟲症。在犬蛔蟲的小鼠模式裡,目前發現犬蛔蟲幼蟲能在受感染並啟動免疫反應的動物宿主體內長時間存活,然而卻無法解釋犬蛔蟲幼蟲逃避宿主的免疫撲殺的機轉為何。
本論文對人體內臟幼蟲移行症之病原犬蛔蟲是否可藉排泌性抗原誘發免疫細胞進行凋亡以逃避宿主的免疫補殺;或發炎細胞是否存在抗凋亡的機制進而導致慢性發炎的可能原因進行探討。
利用不同濃度(5、15、25、50、100 μg/ml)的犬蛔蟲幼蟲排泌性抗原或不同數量的犬蛔蟲幼蟲(150隻、300隻)與human Jurkat T lymphocyte cell line (clone E6-1)進行共同培養12、24、48或72小時後,發現以25 μg/ml 犬蛔蟲幼蟲排泌性抗原與human Jurkat T lymphocyte共同培養24小時後有最佳的致細胞凋亡的效果,然而隨著抗原量及共同培養時間的增加,細胞抗凋亡蛋白分子Bcl-2及Bcl-xL亦有明顯增加的趨勢。此外以300隻的犬蛔蟲幼蟲與human Jurkat T lymphocyte共同培養72小時後有明顯的凋亡表現,因此時cleaved caspase-8有顯著表現。
綜合上述結果推論犬蛔蟲幼蟲入侵宿主後可藉由幼蟲所分泌的排泌性抗原及蟲體本身引致human Jurkat T lymphocyte進行凋亡以逃避宿主的免疫撲殺。
英文摘要 Toxocara canis is one of the most commonly reported zoonotic helminth infections in the world. Humans can get toxocariasis by ingesting infective embryonated eggs. In current studies of T. canis infections using the murine model, researchers found that T. canis larvae could live in paratentic host such as mice for a long time. However, there’s no evidence to prove that T. canis larvae can evade the killing reaction controlled by host immunity.
Here, we intended to investigate whether human visceral larva migrans pathogen Toxocara canis may trigger apoptosis in inflammatory cells by releasing excretory-secretory (TcES) antigens that benefit larval evasion of immunological attacks, or whether there exists anti-apoptosis mechanisms in inflammatory cells that lead to chronic inflammation.
We use different concentrations (5、15、25、50、100 μg/ml) of TcES or different amount (150 or 300) of T. canis larvae co-cultured with human Jurkat T lymphocytes cell line (clone E6-1) for 12~72 hrs to explore the underline mechanism of larval evasion in host as assessed by flow cytometry and western blotting approaches. Our results indicated that 25 μg/ml TcES posed the strongest apoptotic effects on human Jurkat T lymphocyte for 24 hrs co-culture; whereas the expression of anti-apoptotic proteins of Bcl-2 and Bcl-xL increased with the increased concentration of TcES and co-culture time as well. In addition, 300 T. canis larvae are able to trigger apparently apoptosis of human Jurkat T lymphocyte for co-culture of 72 hrs due to enhanced caspase-8 expression.
Based on above findings, we postulate that T. canis larvae may evade immunological attacks by releasing excretory-secretory materials or larvae per se through triggering human Jurkat T lymphocyte apoptosis.
論文目次 縮寫表 ……………………………………………………………… P1
中文摘要 …………………………………………………………… P2
英文摘要 ………………………………………………………… P3~P4
壹、緒論
一、犬蛔蟲(Toxocara canis)的生活史 ………………………… P5
二、人體犬蛔蟲症 ……………………………………………… P6~P7
三、人體犬蛔蟲症之流行病學 ………………………………… P7~P8
四、犬蛔蟲症臨床診斷與治療 ………………………………… P8~P9
五、犬蛔蟲症之實驗動物模式研究與T-lymphocytes於犬蛔蟲症之角色 … P9~P11
六、細胞凋亡 ………………………………………………… P11~P12
貳、研究動機 ………………………………………………… P13~P14
参、研究目的與實驗設計 ………………………………………… P15
肆、實驗方法與步驟
一、犬蛔蟲成蟲採集與感染型蟲卵之取得 ……………………… P16
二、犬蛔蟲感染性幼蟲孵育與排泌性抗原之取得 ………… P16~P17
三、細胞株之取得與培養 ………………………………………… P17
四、犬蛔蟲幼蟲排泌性抗原與Human Jurkat T lymphocytes共同培養 ……… P17
五、犬蛔蟲幼蟲與Human Jurkat T lymphocytes共同培養 …P17~P18
六、 利用Flow cytometry analysis偵測細胞凋亡或抗凋亡的變化 …………… P18
七、利用western blotting檢測細胞凋亡相關的蛋白分子的表現 ………… P18~P19
八、結果統計分析 ………………………………………………… P19
伍、結果
一、犬蛔蟲蟲卵收集與幼蟲孵育 ………………………………… P20
二、以western blotting確認所收集的培養液是否含有犬蛔蟲幼蟲排泌性抗原(TcES) …………………………………………………… P20
三、Flow cytometry偵測TcES與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 ……………………………… P20~P22
四、western blotting偵測TcES與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 …………………………… P22~P23
五、利用western blotting偵測T. canis larvae與human Jurkat T lymphocyte共同培養後細胞凋亡或抗凋亡的變化 ………… P23~P24
陸、討論 ……………………………………………………… P25~P28
柒、參考文獻 ………………………………………………… P29~P40
Figures 1~7 ……………………………………………………P41~P50
Appendix 1~4 ………………………………………………… P51~P54
參考文獻 Ajayi OO, Duhlinska DD, Agwale SM, Njoku M. (2000) Frequency of human toxocariasis in Jos, Plateau State, Nigeria. Mem Inst Oswaldo Cruz. 95, 147-149
Akao N, Tomoda M, Hayashi E, Suzuki R, Shimizu-Suganuma M, Shichinohe K, Fujita K. (2003) Cerebellar ataxia due to Toxocara infection in Mongolian gerbils, Meriones unguiculatus. Vet Parasitol. 113, 229-237
Aldawek AM, Levkut M, Revajova V, Kolodzieyski L, Seveikova Z, Dubinsky P. (2002) Larval toxocarosis in sheep: the immunohistochemical characterization of lesions in some affected organs. Vet Parasitol. 105, 207-214
B. Good, C. V. Holland, M. R. H. Taylor, J. Larragy, P. Moriarty, and M. O’Regan. (2004) Ocular Toxocariasis in Schoolchildren. Clin Infect Dis. 39, 173–178
Beaver PC. (1956) Larva migrans. Exp Parasitol. 5, 587-621
Bachli, H., Minet, J. C. & Gratzl, O. (2004). Cerebral toxocariasis: a possible cause of epileptic seizure in children. Childs Nerv Syst., 63, 301–387.
Blobe GC, Schiemann WP, Lodish HF. (2000) Role of transforming growth factor beta in human disease. N Engl J Med.342, 1350-1358.
Chow SC, Brown A, Pritchard D. (2000) The human hookworm pathogen Necator americanus induces apoptosis in T lymphocytes. Parasite Immunol 22, 21-29.
Davies SJ, Lim KC, Blank RB, Kim JH, Lucas KD, Hernandez DC, Sedgwick JD, McKerrow JH. (2004) Involvement of TNF in limiting liver pathology and promoting parasite survival during schistosome infection. Int J Parasitol. 34, 27-36.
DosReis GA, Ribeiro-Gomes FL, Guillermo LV, Lopes MF. (2007) Cross-talk between apoptosis and cytokines in the regulation of parasitic infection. Cytokine Growth Factor Rev. 18, 97-105.
D'Elia R, Behnke JM, Bradley JE, Else KJ. (2009) Regulatory T cells: a role in the control of helminth-driven intestinal pathology and worm survival. J Immunol. 182, 2340-8.
Epe C, Sabel T, Schnieder T, Stoye M. (1994) The behavior and pathogenicity of Toxocara canis larvae in mice of different strains. Parasitol Res. 80, 691-695.
Fan CK, Lin YH, Du WY, Su KE. (2003) Infectivity and pathogenicity of 14-month-cultured embryonated eggs of Toxocara canis in mice. Vet Parasitol. 113, 145-155.
Fan CK, Hung CC, Du WY, Liao CW, Su KE. (2004a) Seroepidemiology of Toxocara canis infection among mountain aboriginal schoolchildren living in contaminated districts in eastern Taiwan. Trop Med Int Health. 9, 1312-1318.
Fan CK, Hung CC, Lin YH, Li MS, Su KE. (2004b) Enhanced expression of transforming growth factor-beta1 in inflammatory cells and secretory granules in Paneth cells in the small intestine of mice infected with Toxocara canis. Parasitol Res. 94, 397-404.
Fan CK, Lan HS, Hung CC, Chung WC, Liao CW, DU WY, Su KE. (2004c) Seroepidemiology of Toxocara canis infection among mountain aboriginal adults in Taiwan. Trop Med Int Health. 71, 216-221.
Fan CK, Lin YH, Hung CC, Su KE. (2004d) Larval migratory behavior of long-term-maintained Toxocara canis embryonated eggs in mice. Taiwan Vet J 30, 99-105.
Fillaux, J., Santillan, G., Magnaval, J. F., Jensen, O., Larrieu, E. & Sobrino-Becaria, C. D. (2007). Epidemiology of toxocariasis in a steppe environment:the Patagonia study. Am J Trop Med Hyg. 76, 1144–1147.
George A. DosReis, Flavia L. Ribeiro-Gomes, Landi V.C. Guillermo, Marcela F. Lopes. (2007) Cross-talk between apoptosis and cytokines in the regulation of parasitic infection. Cytokine & Growth Factor Rev. 18, 97–105
Glickman LT, Magnaval JF. (1993) Zoonotic roundworm infections. Infect Dis Clin North Am. 7, 717-732.
Glickman LT, Schantz PM, Dombroske R & Cypess R. (1978) Evaluation of serodiagnostic tests for visceral larva migrans. Am J Trop Med Hyg. 27, 492-498.
Gueglio B, de Gentile L, Nguyen JM, Achard J, Chabasse D, Marjolet M. (1994) Epidemiologic approach to human toxocariasis in western France. Parasitol Res. 80, 531-536.
Hakim SL, Mak JW, Lam PL, Nazma ,. Normaznah Y. (1992) Seroprevalence of Toxocara canis antibodies among Orang Asli (aborigines) in Peninsular Malaysia. Southeast Asian J Trop Med Public Health. 23, 493-496.
Hassan AT, el-Manawaty NH. (1994) Experimental murine toxocariasis histopathological study of chronic renal infection, transplacental transmission and ultrastructural study of egg shell. J Egypt Soc Parasitol. 24, 333-339.
Herrmann N, Glickman LT, Schantz PM, Weston MG, Domanski LM. (1985) Seroprevalence of zoonotic toxocariasis in the United States: 1971-1973. Am J Epidemiol. 122, 890-896.
Holland CV, O'Lorcain P, Taylor MR, Kelly A. (1995). Sero-epidemiology of toxocariasis in school children. Parasitol. 110, 535-45.
Hotez, P. J. & Wilkins, P. P. (2009). Toxocariasis: America’s most common neglected infection of poverty and a helminthiasis of global importance? PLoS Negl Trop Dis. 3, e400.
Jimenez JF, Valladares B, Fernandez-Palacios JM, de Armas F, del Castillo A. (1997) A serologic study of human toxocariasis in the Canary Islands (Spain): environmental influences. Am J Trop Med Hyg. 56, 113-115.
Kayes SG. (1997) Human toxocariasis and the visceral larva migrans syndrome: correlative immunopathology. Chem Immunol. 66, 99-124.
Kim KA, Lee YA, Shin MH. (2007) Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica. Int J Parasitol. 37, 1209–1219
Kroemer G & Martin SJ. (2005) Caspase-independent cell death. Nat Med. 11, 725-30
Laura I. Rutitzky, Gerardo A. Mirkin, and Miguel J. Stadecker (2003) Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egg-induced immunopathology in murine schistosomiasis. J Immunol. 171, 1859-67.
Liao CW, Cho WL, Kao TC, Su KE, Lin YH, Fan CK. (2008a) Blood-brain barrier impairment with enhanced SP, NK-1R, GFAP and claudin-5 expressions in experimental cerebral toxocariasis. Parasite Immunol. 30, 525-34.
Liao CW, Fan CK, Kao TC, Ji DD, Su KE, Lin YH, Cho WL. (2008b) Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice. BMC Infect Dis. 8, 84.
Liao CW, Sukati H, D'Lamini P, Chou CM, Liu YH, Huang YC, Chung MH, Mtsetfwa JS, Jonato J, Chiu WT, Chang PW, Du WY, Chan HC, Chu TB, Cheng HC, Su WW, Tu CC, Cheng CY, Fan CK. (2010) Seroprevalence of Toxocara canis infection among children in Swaziland, southern Africa. Ann Trop Med Parasitol.104, 73-80.
Lloyd S, Gemmell MA. (1992) Efficacy of a drug combination of praziquantel, pyrantel embonate, and febantel against helminth infections in dogs. Am J Vet Res 12, 2272-3.
Luder CG, Gross U. (2005) Apoptosis and its modulation during infection with Toxoplasma gondii: molecular mechanisms and role in pathogenesis. Curr Top Microbiol Immunol. 289, 219-237.
Luo ZJ, Wang GX, Yang CI, Luo CH, Cheng SW, Liao L. (1999) Detection of circulating antigens and antibodies in Toxocara canis infection among children in Chengdu, China. J Parasitol. 85, 252-6.
Luo ZJ, Cheng SW, Liao L. (1999) A method for isolation and purification of nematode larvae. J Parasitol. 85, 573-4.
Magnaval J-F, Fabre R, Maurieres P, Charlet J-P, De Larrard B (1991) Application of the western-blotting procedure for the immunodiagnosis of human toxocariasis. Parasitol Res 77, 697-702.
Magnaval J-F, Galindo V, Glickman LT, Clanet M. (1997) Human Toxocara infection of the central nervous system and neurological disorders: a case-control study. Parasitology 115, 537-43.
Magnaval J-F, Glickman LT, Dorchies P, Morassin B. (2001) Highlights of human toxocariasis. Korean J Parasitol. 39, 1-11
Maizels RM, Kennedy MW, Meghji M, Robertson BD, Smith HV. (1987) Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis. J Immunol 139, 207-14.
Mary E. Shawgo, Shary N. Shelton, and John D. Robertson (2008) Caspase-mediated BAk activation and cytochrome c release during intrinsic apoptotic cell deathe in Jurkat cells. J Biol Chem. 283, 35532–35538
Minvielle MC, Niedfeld G, Ciarmela ML, De Falco A, Ghiani H, Basualdo JA. (1999) Asthma and covert toxocariasis. Medicina 59, 243-248.
Moreira-Silva SF, Leao ME, Mendonca HF, Pereira FE. (1998) Prevalence of anti-Toxocara antibodies in a random sample of inpatients at a children's hospital in Vitoria, Espirito Santo, Brazil. Rev Inst Med Trop Sao Paulo 40, 259-261.
Nicoletti, A., Bartoloni, A., Reggio, A., Bartalesi, F., Roselli, M., Sofia, V., Rosado-Chavez, J., Gamboa- Barahona, H., Paradisi, F., Cancrini, G., Tsang, V. C. & Hall, A. J. (2002). Epilepsy, cysticercosis, and toxocariasis: a population-based case–control study in rural Bolivia. Neurol, 58, 1256–1261.
Nicoletti, A., Bartoloni, A., Sofia, V., Mantella, A., Nsengiyumva, G., Frescaline, G. & Preux, P. M. (2007). Epilepsy and toxocariasis: a case–control study in Burundi. Epilepsia, 48, 894–899.
Nicoletti, A., Sofia, V., Mantella, A., Vitale, G., Contrafatto, D., Sorbello, V., Biondi, R., Preux, P. M., Garcia, H. H., Zappia, M. & Bartoloni, A. (2008). Epilepsy and toxocariasis: a case–control study in Italy. Epilepsia, 49, 594–599.
O'Connell KM & Rogan MT. (2000) Apoptosis in human Jurkat T cells after culture with live Taenia crassiceps cysticerci in vitro. Parasitol. 120, 649-55.
Pourgholami MH, Khachigian LM, Fahmy RG, Badar S, Wang L, Chu SW, Morris DL. (2010) Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis. Biochem Biophys Res Commun. doi:10.1016/j.bbrc.2010.06.019
Prestes-Carneiro, L. E., Souza, D. H. P., Moreno, G. C., Troiani, C., Santare´m, V., Zago, S. C. S., Miguel, N. A., Freitas, S. B. Z., Faria, R., Martini, L., Rubinsky-Elefant, G., Iha, A. & Vaz, A. J. (2009). Toxocariasis/cysticercosis seroprevalence in a longtermrural settlement, Saõ Paulo, Brazil. Parasitol, 136, 681–689.
Radman NE, Archelli SM, Fonrouge RD, del V Guardis M, Linzitto OR. (2000) Human toxocarosis. Its seroprevalence in the city of La Plata. Mem Inst Oswaldo Cruz. 95, 281-285.
Robert M. Amthony, Laura I. Rutitzky, Joseph F. Urban Jr., Miguel J. Seadecker, and William C. Gause. (2007) Protective immune mechanism in helminth infection. Nat Rev Immunol. 7, 975-987
Rodman J. & Pizzimenti J., (2009). In vivo diagnostic imaging of ocular toxocariasis. Clin Exp Optom. 92, 146-9
Rolda´n, W. H., Espinoza, Y. A., Huapaya, P. E., Huiza, A. F., Sevilla, C. R. & Jime´nez, S. (2009). Frequency of human toxocariasis in a rural population from Cajamarca, Peru determined by DOT-ELISA test. Rev Inst Med Trop Sao Paulo. 51, 67–71.
Roshanak Tolouei Semnani, Priyanka Goel Venugopal, Lily Mahapatra, Jason A. Skinner, Francoise Meylan, Daniel Chien, David W. Dorward, Damien Chaussabel, Richard M. Siegel and Thomas B. Nutman (2008) Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi. J Immunol. 181, 7081-9.
Roshanak Tolouei Semnani, Anne Y. Liu, Helen Sabzevari, Joseph Kubofcik, Jun Zhou, Julia K. Gilden and Thomas B. Nutman. (2010) Brugia malayi microfilariae induce cell death in human dendritic cells, inhibit their ability to make IL-12 and IL-10, and reduce their capacity to activate CD4+ T cells. J Immunol 171, 1950-60.
Rubinsky-Elefant G, Hirata CE, Yamamoto JH, Ferreira MU. (2010) Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms. Ann Trop Med Parasitol 104, 3-23.
Rutitzky LI. Mirkin GA, Stadecker MJ. (2003) Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egg-induced immunopathology in murine schistosomiasis. J Immunol. 171, 1859-67.
Semnani R.T., Liu AY, Sabzevari H, Kubofcik J, Zhou J, Gilden JK, Nutman TB., (2003) Brugia malayi microfilariae induce cell death in human dendritic cells, inhibit their ability to make IL-12 and IL-10, and reduce their capacity to activate CD4+ T cells. J Immunol. 171, 1950-60.
Semnani R.T., Venugopal PG, Mahapatra L, Skinner JA, Meylan F, Chien D, Dorward DW, Chaussabel D, Siegel RM, Nutman TB., (2008) Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi. J Immunol. 181, 7081-9.
Sharif M, Daryani A, Barzegar G, Nasrolahei M, Khalilian A. (2010) Seroprevalence of toxocariasis in schoolchildren in Northern Iran. Pak J Biol Sci. 13, 180-4.
Smith H, Holland C, Taylor M, Magnaval JF, Schantz P, Maizels R. (2009) How common is human toxocariasis? Towards standardizing our knowledge. Trends in Parasitology 25, 182-188.
Solano S, Cortés IM, Copitin NI, Tato P, Molinari JL. (2006) Lymphocyte apoptosis in the inflammatory reaction around Taenia solium metacestodes in porcine cysticercosis. Vet Parasitol. 31, 171-6.
Stensvold, C. R., Skov, J., Moller, L. N. Jensen, P. M., Kapel, C. M. O., Petersen, E. & Nielsen, H. V. (2009). Seroprevalence of human toxocariasis in Denmark. Clin Vaccine Immunol 16, 1372–1373.
Takamoto, M.; Wang, Z.X.; Watanabe, N.; Matsuzawa, A.; Nariuchi, H.; Sugane, K., (1998) Eosinophilia, IgE production, and cytokine production by lung T cells in surface CD4-deficient mutant mice infected with Toxocara canis. Immunol 95, 97-104.
Tato P, Fernandez AM, Solano S, Borgonio V, Garrido E, Sepulveda J, Molinari JL. (2004) A cysteine protease from Taenia solium metacestodes induce apoptosis in human CD4+ T-cells. Parasitol Res 92,197-204.
Uga S, Matsumura T, Fujisawa K, Okubo K, Kataoka N, Kondo K. (1990) Incidence of seropositivity to human toxocariasis in Hyogo prefecture Japan and its possible role in opthalmic disease. Japanese J Parasitol 39, 500-502.
Urban JP, Douvres FW, Trmba FG. (1981) A rapidmethod for hatching Ascaris suum eggs in vitro. Helminthol. Society of Washinton 48, 241-243.
Watthanakulpanich D, Smith HV, Hobbs G, Whalley AJ, Billington D. (2008) Application of Toxocara canis excretory-secretory antigens and IgG subclass antibodies (IgG1-4) in serodiagnostic assays of human toxocariasis. Acta Trop. 106, 90-5
Won, K. Y., Kruszon-Moran, D., Schantz, P. M. & Jones, J. L. (2008). National seroprevalence and risk factors for zoonotic Toxocara spp. infection. Am J Trop Med Hyg. 79, 552–557.

------------------------------------------------------------------------ 第 12 筆 ---------------------------------------------------------------------
系統識別號 U0007-0308201013434900
論文名稱(中文) 腦神經滋養因子濃度與生物節律基因於酒癮患者有無併發譫妄現象在血中之呈現與差異
論文名稱(英文) Differential Patterns of Serum Brain-Derived Neurotrophic Factor Levels and Circadian Rhythm Gene Expression in Alcoholic Patients with and without Delirium Tremens During Acute Withdrawal
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 黃名琪
學號 D105095003
學位類別 博士
語文別 中文
口試日期 2010-07-26
論文頁數 70頁
口試委員 指導教授-呂思潔
委員-李怡萱
委員-藍先元
委員-王家儀
委員-劉興政
關鍵字(中) 腦神經滋養因子
生物節律
生物節律基因
酒精依賴, 酒癮, 酒精戒斷, 譫妄
關鍵字(英) brain-derived neurotrophic factor
BDNF
circadian rhythm
circadian clock gene
alcohol dependence
alcohol withdrawal
delirium tremens
alcohol withdrawal delirium
學科別分類
中文摘要 背景: 物質戒斷係與成癮現象有密切相關,探討酒精戒斷時期相關之神經毒性與神經適應性,可協助了解酒精之成癮機轉。震顫性譫妄乃為酒精戒斷症候群中最嚴重的表現,目前認為具有震顫性譫妄的個案因其較差預後而被視為一特異族群。腦神經滋養因子(Brain-derived neurotrophic factor,BDNF)在成癮發展過程之神經塑性與學習扮演重要角色,同時,目前證據顯示長期飲酒和生物節律異常有明顯相關,生物節律乃由數個生物節律基因所調控,包括hClock1, hBmal1, hPer1, hPer2, hCry1, hCry2。然而有關長期飲酒導致生物節律基因的表現改變,多以動物研究呈現,尚未有人體證據。因此,我們將探討酒癮患者血中的BDNF濃度表現以及周邊淋巴球的生物節律基因mRNA表現量,以及在停酒一週後,亦即早期戒斷後之表現,同時比較患者有無併發譫妄現象在這兩個指標上的差別表現。並且探討和併發譫妄之關聯。
方法: 65位符合精神科診斷準則第四版中酒精依賴的住院患者,與39位健康控制組加入研究。患者在住院治療期間依其呈現譫妄現象與否而分組為譫妄組(25位)與非譫妄組(40位)。所有的個案在早上9到10點間接受抽血,但酒癮者則抽血兩個時間點,亦即住院隔天早上一次(基準值)與住院第七天(第一週)。血清中BDNF濃度則由sandwich enzyme-linked immunosorbent assay (ELISA)檢測。其中,我們由全血中分離並收集22 位男位酒精依賴患者與12位控制的周邊淋巴球,以定量real-time PCR測定hClock1, hBmal1, hPer1, hPer2, hCry1, hCry2 的基因表現量。
結果: 血清中BDNF濃度在控制組、非譫妄組、譫妄組在統計上呈現顯著的組間差異,分別是14.8 ± 4.7 ng/mL, 12.3 ± 3.3 ng/mL與6.2 ± 2.6 ng/mL (p < 0.001). 在一週的戒斷後,酒精依賴患者的兩組血清中BDNF濃度有顯著增加,雖然非譫妄組的BDNF濃度已和控制組無差異(13.4 ± 3.5 ng/mL),但譫妄組個案則仍然顯著低於控制組 (8.9 ± 4.4 ng/mL)。至於生物節律基因的表現量方面, 兩組酒精依賴個案均遠低於控制組個案(p < 0.001),即使經過一週之戒斷時間,兩組酒精依賴個案生物節律基因mRNA表現仍少有改善,並且有無譫妄發生僅有小幅影響兩組之間的表現差異。
討論: 我們的研究指出長期飲酒會造成酒精依賴患者體內的BDNF濃度下降,同時我們發現具有更為低下的BDNF濃度和併發譫妄可能有關。此外,適當的戒斷治療會協助BDNF濃度的回復。這指出BDNF 可能與酒精戒斷症候群中不同表現型有關,並參與相關神經適應性的調控。但我們卻發現無論有無併發譫妄,幾乎每個指標生物節律基因表現量在所有酒精依賴個案的皆呈嚴重不足,這個現象並且在戒斷一週後並無太大改變。雖然本研究呈現人體長期飲酒後生物節律基因表現量變化的首次發表,但我們的檢測為單點研究,未來仍需佐以其他生物節律指標的資料,以提供酒精破壞生物節律型態更為直接有力的證據。由於 BDNF也參與生物節律基因的調控,從而影響神經塑性,所以亦將進一步探討其與指標生物節律基因表現量之相關性。
英文摘要 Background: Alcohol withdrawal-enhanced neuroadaptation contributes to the addictive process. Delirium tremens (DTs) is the most serious complication of alcohol withdrawal syndrome (AWS) and postulated to be a clinically distinct phenotype among AWS. Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity and learning related to addiction. Meanwhile, current evidence indicated a clear interaction between chronic alcohol consumption and disrupted circadian rhythmicity which is regulated by several circadian clock genes including hClock1, hBmal1, hPer1, hPer2, hCry1, hCry2. Studies exploring the altered expressions of these genes in alcohol addiction have been mainly described in animals. Therefore, we assessed the differences in serum BDNF levels as well as the mRNA expression of circadian clock genes, measured at baseline and one week after alcohol withdrawal among alcoholic patients with and without DT.
Methods: Sixty-five inpatients, fulfilling the DSM-IV criteria of alcohol dependence and admitted for alcohol detoxification, as well as 39 healthy control subjects were enrolled. The alcoholic patients were further divided by the appearance of DTs into the DT group (n = 25) and non-DT group (n = 40). All the participants received blood withdrawal at 9-10 a.m. while the AD patients had blood collection for twice: on the next morning of admission (baseline) and on the 7th day. Among them, the PBMCs of 22 male alcoholics and 12 comparison control subjects were collected from the whole blood. Serum BDNF levels were measured by sandwich enzyme-linked immunosorbent assay while the mRNA expression profiles of hClock1, hBmal1, hPer1, hPer2, hCry1, hCry2 in PBMCs were determined by quantitative real-time PCR.
Results: Serum BDNF levels differed significantly among the three groups: (1) control group 14.8 ± 4.7 ng/mL; (2) non-DT group 12.3 ± 3.3 ng/mL; (3) DT group 6.2 ± 2.6 ng/mL (p < 0.001). After one week after alcohol withdrawal, BDNF levels increased significantly for both alcoholic groups. While non-DT group had comparable BDNF levels (13.4 ± 3.5 ng/mL) with controls, the DT group still exhibited lower levels (8.9 ± 4.4 ng/mL). Regarding to the expression of circadian clock genes, baseline mRNA levels were markedly lower in AD patients than in control subjects. After one week of alcohol detoxification, there were very limited restorations of discrete circadian gene expressions. DT group did not differ in the expression patterns of circadian clock genes from non-DT group.
Conclusions: The present study suggests chronic drinking leads to a reduction in BDNF levels and patients with more deficient BDNF expression are vulnerable to the development of DTs. BDNF levels elevated after prompt alcohol detoxification treatment. Therefore BDNF could involve modifying the phenotypes of AWS as well as the pertinent neuroadaptive processes of AD. In addition, we first demonstrated the overall lowering of circadian clock genes among AD patients. But, the expression pattern is comparable between patients with and without DTs. Though preliminary with data at only one single time point, the observation of strikingly reduced mRNA levels supports the association between circadian clock gene dysregulation and chronic alcohol intake.
論文目次 頁數
中文摘要..………………………………………………………………………….1~2
(Abstract in Chinese)
英文摘要..………………………………………………………………………….3~5
(Abstract in English)
緒論………………………………………………………………………………...6-13
(Introduction)
研究目的…………………………………………………………………………......14
(Study Aims)
研究材料與方法…………………………………………………………………15~20
(Materials and Methods)
實驗結果…………………………………………………………………………21~25
(Results)
討論………………………………………………………………………………26~36
(Discussion)
結論與展望………………………………………………………………………37~38
(Conclusion and Perspective)
參考文獻…………………………………………………………………………39~48
(References)
圖表………………………………………………………………………………49~70
(Tables and Figures)
Table 1. Demographic characteristics and Chinese version of Delirium Rating Scale-Revised-98 (DRS-R-98-C) scores of four diagnostic patients. …..49

Table 2. Internal consistency of Chinese version of Delirium Rating Scale-Revised-98 (DRS-R-98-C) scale in 28 delirious patients. …..50
Table 3. Cutoffs, Sensitivity, and specificity of Chinese version of Delirium Rating Scale-Revised-98 (DRS-R-98-C) when comparing delirious group with all non-delirious groups, based on Receiver Operating Characteristic analysis. …..52
Table 4. Baseline demographic characteristics and biological parameters in control subjects and alcoholic patients. …..53
Table 5. The demographic and clinical characteristics and laboratory data in healthy controls and patients with alcohol dependence (AD) divided by the appearance of DTs (delirium tremens). …..55
Table 6. Target genes, symbols, assay identifications numbers (ID), reference sequence (accession numbers). …..56
Table 7. The comparison of hClock1, hBmal1, hPer1, hPer2, hCry1, hCry2 mRNA expressions in two AD groups and healthy controls at baseline as well as the comparison in two AD groups one week after detoxification (Week 1). …..57
Fig. 1. The distribution of Chinese version of Delirium Rating Scale- Revised-98 (DRS-R-98-C) severity and total scores in quartiles (middle 50% in the box) and median scores (in solid black lines) of four patient groups. …..58
Fig. 2. Regression plot between Chinese version of Delirium Rating Scale-Revised-98 (DRS-R-98-C) total scores and Mini-Mental State Examination (MMSE) in 28 delirious patients. …..59
Fig. 3. Serum BDNF levels (mean ± SD) of alcohol patients (n = 25) at baseline and after one-week alcohol withdrawal as well as of the healthy controls (n = 22). ….60
Fig. 4. The correlation between baseline serum BDNF levels and the average of first-day CIWA-Ar scores in alcoholic patients. …..61
Fig. 5. Comparison of the baseline serum BDNF levels between groups (gray) and the changes after one week of detoxification among alcoholic patients (shadowed). …..62
Appendix 1. Chinese version of Clinical Institute Withdrawal Assessment Scale for Alcohol, Revised (CIWA-Ar-C). …..63
Appendix 2. Chinese version of Delirium Rating Scale-R-98. …..66
參考文獻 Abarca C, Albrecht U, Spanagel R (2002) Cocaine sensitization and reward are under the influence of circadian genes and rhythm. Proc Natl Acad Sci U S A 99:9026-9030.
Aloe L, Tuveri MA, Guerra G, Pinna L, Tirassa P, Micera A, Alleva E (1996) Changes in human plasma nerve growth factor level after chronic alcohol consumption and withdrawal. Alcohol Clin Exp Res 20:462-465.
Barnard AR, Nolan PM (2008) When clocks go bad: neurobehavioural consequences of disrupted circadian timing. PLoS Genet 4:e1000040.
Berggren U, Fahlke C, Berglund KJ, Blennow K, Zetterberg H, Balldin J (2009) Thrombocytopenia in early alcohol withdrawal is associated with development of delirium tremens or seizures. Alcohol Alcohol 44:382-386.
Bison S, Crews F (2003) Alcohol withdrawal increases neuropeptide Y immunoreactivity in rat brain. Alcohol Clin Exp Res 27:
Boivin DB, James FO, Wu A, Cho-Park PF, Xiong H, Sun ZS (2003) Circadian clock genes oscillate in human peripheral blood mononuclear cells. Blood 102:4143-4145.
Brower KJ (2001) Alcohol's effects on sleep in alcoholics. Alcohol Res Health 25:110-125.
Brower KJ (2003) Insomnia, alcoholism and relapse. Sleep Med Rev 7:523-539.
Brown ME, Anton RF, Malcolm R, Ballenger JC (1988) Alcohol detoxification and withdrawal seizures: clinical support for a kindling hypothesis. Biol Psychiatry 23:507-514.
Brown SA, Fleury-Olela F, Nagoshi E, Hauser C, Juge C, Meier CA, Chicheportiche R, Dayer JM, Albrecht U, Schibler U (2005) The period length of fibroblast circadian gene expression varies widely among human individuals. PLoS Biol 3:e338.
Castren E, Rantamaki T (2010) The role of BDNF and its receptors in depression and antidepressant drug action: Reactivation of developmental plasticity. Dev Neurobiol 70:289-297.
Chen CP, Kuhn P, Advis JP, Sarkar DK (2004) Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat. J Neurochem 88:1547-1554.
Crews FT, Waage HG, Wilkie MB, Lauder JM (1999) Ethanol pretreatment enhances NMDA excitotoxicity in biogenic amine neurons: protection by brain derived neurotrophic factor. Alcohol Clin Exp Res 23:1834-1842.
Drummond SP, Gillin JC, Smith TL, DeModena A (1998) The sleep of abstinent pure primary alcoholic patients: natural course and relationship to relapse. Alcohol Clin Exp Res 22:1796-1802.
Fiellin DA, O'Connor PG, Holmboe ES, Horwitz RI (2002) Risk for delirium tremens in patients with alcohol withdrawal syndrome. Subst Abus 23:83-94.
Fujimura H, Altar CA, Chen R, Nakamura T, Nakahashi T, Kambayashi J, Sun B, Tandon NN (2002) Brain-derived neurotrophic factor is stored in human platelets and released by agonist stimulation. Thromb Haemost 87:728-734.
Gillin JC, Smith TL, Irwin M, Butters N, Demodena A, Schuckit M (1994) Increased pressure for rapid eye movement sleep at time of hospital admission predicts relapse in nondepressed patients with primary alcoholism at 3-month follow-up. Arch Gen Psychiatry 51:189-197.
Goggi J, Pullar IA, Carney SL, Bradford HF (2002) Modulation of neurotransmitter release induced by brain-derived neurotrophic factor in rat brain striatal slices in vitro. Brain Res 941:34-42.
Gorwood P, Limosin F, Batel P, Hamon M, Ades J, Boni C (2003) The A9 allele of the dopamine transporter gene is associated with delirium tremens and alcohol-withdrawal seizure. Biol Psychiatry 53:85-92.
Guillin O, Diaz J, Carroll P, Griffon N, Schwartz JC, Sokoloff P (2001) BDNF controls dopamine D3 receptor expression and triggers behavioural sensitization. Nature 411:86-89.
Hall FS, Drgonova J, Goeb M, Uhl GR (2003) Reduced behavioral effects of cocaine in heterozygous brain-derived neurotrophic factor (BDNF) knockout mice. Neuropsychopharmacology 28:1485-1490.
Hastings MH, Reddy AB, Maywood ES (2003) A clockwork web: circadian timing in brain and periphery, in health and disease. Nat Rev Neurosci 4:649-661.
Haynes LE, Barber D, Mitchell IJ (2004) Brain Res. Chronic antidepressant medication attenuates dexamethasone-induced neuronal death and sublethal neuronal damage in the hippocampus and striatum. 1026:157-167.
Heberlein A, Bleich S, Bayerlein K, Frieling H, Groschl M, Kornhuber J, Hillemacher T (2008) NGF plasma levels increase due to alcohol intoxication and decrease during withdrawal. Psychoneuroendocrinology 33:999-1003.
Hemmingsen R, Kramp P (1988) Delirium tremens and related clinical states: psychopathology, cerebral pathophysiology and psychochemistry: a two-component hypothesis concerning etiology and pathogenesis. Acta Psychiatr Scand Suppl 345:94-107.
Hensler JG, Ladenheim EE, Lyons WE (2003) Ethanol consumption and serotonin-1A (5-HT1A) receptor function in heterozygous BDNF (+/-) mice. J Neurochem 85:1139-1147.
Hersh D, Kranzler HR, Meyer RE (1997) Persistent delirium following cessation of heavy alcohol consumption: diagnostic and treatment implications. Am J Psychiatry 154:846-851.
Hiller-Sturmhofel S, Kulkosky P (2001) Chronobiological regulation of alcohol intake. Alcohol Res Health 25:141-148.
Horger BA, Iyasere CA, Berhow MT, Messer CJ, Nestler EJ, Taylor JR (1999) Enhancement of locomotor activity and conditioned reward to cocaine by brain-derived neurotrophic factor. J Neurosci 19:4110-4122.
Huang MC, Chen CH, Liu SC, Ho CJ, Shen WW, Leu SJ (2008) Alterations of serum brain-derived neurotrophic factor levels in early alcohol withdrawal. Alcohol Alcohol 43:241-245.
Huang MC, Lee CH, Lai YC, Kao YF, Lin HY, Chen CH (2009) Chinese version of the Delirium Rating Scale-Revised-98: reliability and validity. Compr Psychiatry 50:81-85.
Iijima M, Nikaido T, Akiyama M, Moriya T, Shibata S (2002) Methamphetamine-induced, suprachiasmatic nucleus-independent circadian rhythms of activity and mPer gene expression in the striatum of the mouse. Eur J Neurosci 16:921-929.
Imatoh N, Nakazawa Y, Ohshima H, Ishibashi M, Yokoyama T (1986) Circadian rhythm of REM sleep of chronic alcoholics during alcohol withdrawal. Drug Alcohol Depend 18:77-85.
James FO, Boivin DB, Charbonneau S, Belanger V, Cermakian N (2007) Expression of clock genes in human peripheral blood mononuclear cells throughout the sleep/wake and circadian cycles. Chronobiol Int 24:1009-1034.
Jockers-Scherubl MC, Bauer A, Kuhn S, Reischies F, Danker-Hopfe H, Schmidt LG, Rentzsch J, Hellweg R (2007) Nerve growth factor in serum is a marker of the stage of alcohol disease. Neurosci Lett 419:78-82.
Joe KH, Kim YK, Kim TS, Roh SW, Choi SW, Kim YB, Lee HJ, Kim DJ (2007) Decreased plasma brain-derived neurotrophic factor levels in patients with alcohol dependence. Alcohol Clin Exp Res 31:1833-1838.
Karege F, Bondolfi G, Gervasoni N, Schwald M, Aubry JM, Bertschy G (2005) Low brain-derived neurotrophic factor (BDNF) levels in serum of depressed patients probably results from lowered platelet BDNF release unrelated to platelet reactivity. Biol Psychiatry 57:1068-1072.
Karege F, Schwald M, Cisse M (2002) Postnatal developmental profile of brain-derived neurotrophic factor in rat brain and platelets. Neurosci Lett 328:261-264.
Kim TS, Kim DJ, Lee H, Kim YK (2007) Increased plasma brain-derived neurotrophic factor levels in chronic smokers following unaided smoking cessation Neurosci Lett 423:53-57.
Kodama H, Nakazawa Y, Kotorii T, Nonaka K, Inanaga K, Ohshima M, Tokoyama T (1988) Biorhythm of core temperature in depressive and non-depressive alcoholics. Drug Alcohol Depend 21:1-6.
Koob GF (2003) Alcoholism: allostasis and beyond. Alcohol Clin Exp Res 27:232-243.
Kovacs GL (2003) Natriuretic peptides in alcohol withdrawal: central and peripheral mechanisms. Curr Med Chem 10:2559-2576.
Kuhlwein E, Hauger RL, Irwin MR (2003) Abnormal nocturnal melatonin secretion and disordered sleep in abstinent alcoholics. Biol Psychiatry 54:1437-1443.
Kusanagi H, Hida A, Satoh K, Echizenya M, Shimizu T, Pendergast JS, Yamazaki S, Mishima K (2008) Expression profiles of 10 circadian clock genes in human peripheral blood mononuclear cells. Neurosci Res 61:136-142.
Lewin GR, Barde YA (1996) Physiology of the neurotrophins. Annu Rev Neurosci 19:289-317.
Lindvall O, Kokaia Z, Bengzon J, Elmer E, Kokaia M (1994) Neurotrophins and brain insults. Trends Neurosci 17:490-496.
Liu Y, Wang Y, Wan C, Zhou W, Peng T, Wang Z, Li G, Cornelisson G, Halberg F (2005) The role of mPer1 in morphine dependence in mice. Neuroscience 130:383-388.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.
Lommatzsch M, Braun A, Mannsfeldt A, Botchkarev VA, Botchkareva NV, Paus R, Fischer A, Lewin GR, Renz H (1999) Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived Neurotrophic functions. Am J Pathol 155:1183-1193.
MacLennan AJ, Lee N, Walker DW (1995) Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus. Neurosci Lett 197:105-108.
Matsushita S, Kimura M, Miyakawa T, Yoshino A, Murayama M, Masaki T, Higuchi S (2004) Association study of brain-derived neurotrophic factor gene polymorphism and alcoholism. Alcohol Clin Exp Res 28:1609-1612.
Mayo-Smith MF (1997) Pharmacological management of alcohol withdrawal. A meta-analysis and evidence-based practice guideline. American Society of Addiction Medicine Working Group on Pharmacological Management of Alcohol Withdrawal. JAMA 278:144-151.
McClung CA, Sidiropoulou K, Vitaterna M, Takahashi JS, White FJ, Cooper DC, Nestler EJ (2005) Regulation of dopaminergic transmission and cocaine reward by the Clock gene. Proc Natl Acad Sci U S A 102:9377-9381.
McGough NN, He DY, Logrip ML, Jeanblanc J, Phamluong K, Luong K, Kharazia V, Janak PH, Ron D (2004) RACK1 and brain-derived neurotrophic factor: a homeostatic pathway that regulates alcohol addiction. J Neurosci 24:10542-10552.
McKeon A, Frye MA, Delanty N (2008) The alcohol withdrawal syndrome. J Neurol Neurosurg Psychiatry 79:854-862.
Miller MW (2004) Repeated episodic exposure to ethanol affects neurotrophin content in the forebrain of the mature rat. Exp Neurol 189:173-181.
Mukai M, Uchimura N, Hirano T, Ohshima H, Ohshima M, Nakamura J (1998) Circadian rhythms of hormone concentrations in alcohol withdrawal. Psychiatry Clin Neurosci 52:238-240.
Nestler EJ (2001) Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2:119-128.
Nixon K, Crews FT (2004) Temporally specific burst in cell proliferation increases hippocampal neurogenesis in protracted abstinence from alcohol. J Neurosci 24:9714-9722.
Pallier PN, Maywood ES, Zheng Z, Chesham JE, Inyushkin AN, Dyball R, Hastings MH, Morton AJ (2007) Pharmacological imposition of sleep slows cognitive decline and reverses dysregulation of circadian gene expression in a transgenic mouse model of Huntington's disease. J Neurosci 27:7869-7878.
Palmstierna T (2001) A model for predicting alcohol withdrawal delirium. Psychiatr Serv 52:820-823.
Pan W, Banks WA, Fasold MB, Bluth J, Kastin AJ (1998) Transport of brain-derived neurotrophic factor across the blood-brain barrier. Neuropharmacology 37:1553-1561.
Perreau-Lenz S, Zghoul T, de Fonseca FR, Spanagel R, Bilbao A (2009) Circadian regulation of central ethanol sensitivity by the mPer2 gene. Addict Biol 14:253-259.
Poduslo JF, Curran GL (1996) Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF. Brain Res Mol Brain Res 36:280-286.
Radka SF, Holst PA, Fritsche M, Altar CA (1996a) Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay. Brain Res 709:122-301.
Radka SF, Holst PA, Fritsche M, Altar CA (1996b) Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay. Brain Res 709:122-301.
Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418:935-941.
Rosenwasser AM (2001) Alcohol, antidepressants, and circadian rhythms. Human and animal models. Alcohol Res Health 25:126-135.
Rosenwasser AM, Fecteau ME, Logan RW (2005) Effects of ethanol intake and ethanol withdrawal on free-running circadian activity rhythms in rats. Physiol Behav 84:537-542.
Sano H, Suzuki Y, Yazaki R, Tamefusa K, Ohara K, Yokoyama T, Miyasato K, Ohara K (1993) Circadian variation in plasma 5-hydroxyindoleacetic acid level during and after alcohol withdrawal: phase advances in alcoholic patients compared with normal subjects. Acta Psychiatr Scand 87:291-296.
Schmitz MM, Sepandj A, Pichler PM, Rudas S (1996) Disrupted melatonin-secretion during alcohol withdrawal. Prog Neuropsychopharmacol Biol Psychiatry 20:983-995.
Segal M, Avital A, Rusakov A, Sandbank S, Weizman A (2009) Serum creatine kinase activity differentiates alcohol syndromes of dependence, withdrawal and delirium tremens. Eur Neuropsychopharmacol 19:92-96.
Spanagel R, Pendyala G, Abarca C, Zghoul T, Sanchis-Segura C, Magnone MC, Lascorz J, Depner M, Holzberg D, Soyka M, Schreiber S, Matsuda F, Lathrop M, Schumann G, Albrecht U (2005a) The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med 11:35-42.
Spanagel R, Rosenwasser AM, Schumann G, Sarkar DK (2005b) Alcohol consumption and the body's biological clock. Alcohol Clin Exp Res 29:1550-1557.
Swanwick CC, Murthy NR, Kapur J (2006) Activity-dependent scaling of GABAergic synapse strength is regulated by brain-derived neurotrophic factor. Mol Cell Neurosci 31:481-492.
Tapia-Arancibia L, Rage F, Givalois L, Dingeon P, Arancibia S, Beauge F (2001) Effects of alcohol on brain-derived neurotrophic factor mRNA expression in discrete regions of the rat hippocampus and hypothalamus. J Neurosci Res 63:200-208.
Teboul M, Barrat-Petit MA, Li XM, Claustrat B, Formento JL, Delaunay F, Levi F, Milano G (2005) Atypical patterns of circadian clock gene expression in human peripheral blood mononuclear cells. J Mol Med 83:693-699.
Thoenen H (1995) Neurotrophins and neuronal plasticity. Science 270:593-598.
Tsai G, Coyle JT (1998a) The role of glutamatergic neurotransmission in the pathophysiology of alcoholism. Annu Rev Med 49:173-184.
Tsai G, Coyle JT (1998b) The role of glutamatergic neurotransmission in the pathophysiology of alcoholism. Annu Rev Med 49:173-184.
Uz T, Ahmed R, Akhisaroglu M, Kurtuncu M, Imbesi M, Dirim Arslan A, Manev H (2005) Effect of fluoxetine and cocaine on the expression of clock genes in the mouse hippocampus and striatum. Neuroscience 134:1309-1316.
van Munster BC, Korevaar JC, de Rooij SE, Levi M, Zwinderman AH (2007) Genetic polymorphisms related to delirium tremens: a systematic review. Alcohol Clin Exp Res 31:177-184.
Wasielewski JA, Holloway FA (2001) Alcohol's interactions with circadian rhythms. A focus on body temperature. Alcohol Res Health 25:94-100.
Wright T, Myrick H, Henderson S, Peters H, Malcolm R (2006) Risk factors for delirium tremens: a retrospective chart review. Am J Addict 15:213-219.
Yamamoto H, Gurney ME (1990) Human platelets contain brain-derived neurotrophic factor. J Neurosci 10:3469-3478.
Yokota SI, Horikawa K, Akiyama M, Moriya T, Ebihara S, Komuro G, Ohta T, Shibata S (2000) Inhibitory action of brotizolam on circadian and light-induced per1 and per2 expression in the hamster suprachiasmatic nucleus. Br J Pharmacol 131:1739-1747.
Yuferov V, Nielsen D, Butelman E, Kreek MJ (2005) Microarray studies of psychostimulant-induced changes in gene expression. Addict Biol 10:101-118.



------------------------------------------------------------------------ 第 13 筆 ---------------------------------------------------------------------
系統識別號 U0007-0308201014591600
論文名稱(中文) 探討黃酮類化合物對蝕骨細胞與造骨細胞生成的影響
論文名稱(英文) The effects of flavones on the formation of osteoblasts and osteoclasts
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 林鈺翔
學號 M105095009
學位類別 碩士
語文別 中文
口試日期 2010-07-05
論文頁數 70頁
口試委員 指導教授-陳彥州
共同指導教授-蔡郁惠
委員-張恆雄
委員-林鈺玲
委員-葉添順
關鍵字(中) 黃酮素
造骨細胞
蝕骨細胞
關鍵字(英) flavone
osteoblast
osteoclast
學科別分類
中文摘要 中文摘要
骨骼在人體中的功能,包含身體運動、支撐身體、保護臟器等功能。骨骼內人體中並非靜止不變,而是不斷地進行骨分解與骨生成,維持骨質的動態平衡,此過程稱之為骨質重塑(bone remodling)。當造骨細胞進行骨生成時,蝕骨細胞也進行骨分解。當骨分解大於骨生成時,會使骨質流失而導致骨質疏鬆。骨質疏鬆症(osteoporosis)是一種骨骼代謝性疾病,有此疾病者,骨密度降低,骨骼空隙變大,骨骼變得脆弱,容易因外力而造成骨折。婦女停經後易得骨質疏鬆症,乃因缺乏雌性激素所致。在相關研究中發現,給予停經後婦女雌激素有助於改善或預防骨質疏鬆症。臨床上稱此方法為賀爾蒙替代療法(hormone replacement therapy)。但是另有文獻指出補充雌激素會增加乳癌與子宮內膜癌發生的機率。在尋求雌激素之替代研究中發現,有些類黃酮類化合物(flavonoids)有增加骨密度,及減緩骨質流失的功能。故本研究欲探討黃酮類化合物(flavones)對骨骼的影響,我們利用與骨骼代謝有關的兩種細胞:造骨細胞及蝕骨細胞,觀察黃酮類化合物(flavones)對造骨細胞及蝕骨細胞生成的影響。
在實驗中我們觀察黃酮類化合物 (含Baicalein、flavone、6-hydroxy flavone、7-hydroxy flavone、5-methoxy flavone、7-methoxy flavone、luteolin)對MBMM?眵茩M(MCSF dependent bone marrow macrophage)的影響;先以不同濃度的flavones處理前蝕骨細胞兩天後,再加入RANKL與MCSF培養三天來誘導其分化為蝕骨細胞。經TRAP染色後,再數具多核細胞(N≧3)且表現TRAP活性的細胞數量,另一方面也測量這些所細胞分泌出的TRAP之活性。發現這些黃酮類化合物(Baicalein、6-hydroxy flavone、7-hydroxy flavone、5-methoxy flavone、7-methoxy flavone、luteolin)確實有抑制MBMM?眵茩M分化成蝕骨細胞的現象。
另外我們也利用前造骨細胞(MC3T3 cell),來觀察這些黃酮類化合物 (Baicalein、flavone、6-hydroxy flavone、7-hydroxy flavone、5-methoxy flavone、7-methoxy flavone、luteolin)對前造骨細胞骨化的影響。先以不同濃度的黃酮類化合物來處理滿盤之MC3T3細胞4天,再測細胞內ALP的酵素活性,結果發現只有7-methoxy flavone、6-hydroxy flavone會促進造骨細胞的分化。並且7-methoxy flavone會促進造骨細胞的鈣沉積作用。本論文實驗證實,所研究之黃酮類化合物中只有7-methoxy flavone及6-hydroxy flavone同時可促進造骨細胞的成熟並抑制蝕骨細胞生成的,因此,這兩種黃酮類化合物具有預防或治療骨質疏鬆症的最佳潛力。
英文摘要 Abstract
The bone provides mobility, support, and protection of the body. Bone tissue is dynamic;healthy bones require continuous formation and resorption to regulate mineral homeostasis. An imbalance in bone formation and bone resorption result in osteoporosis. Osteoporosis is a kind of metabolic bone disease characterized by reduced bone mass density and increased risk of fractures. Aging and postmenopause are among the most important risk factors for developing osteoporosis. Hormone replacement therapy (HRT) can increase bone mass of menopausal and postmenopausal women. However, hormone replacement therapy (estrogen) also reported to increase the risk of breast cancer. Alternatively, flavonoids are known to have phytoestrogenic effects:increase bone mass and reduce bone loss .
Flavonoids includes Flavones、flavonols、flavanones、catechins、anthocyanidins and Isoflavones. In this study, it was intended to investigate the effects of flavones on osteoblast and osteoclast formation. Seven flavones ( including baicalein、flavone、5-mthoxyflavone、7-mthoxyflavone、6-hydroxyflavone、7-hydroxyflavone and luteolin) were studied on osteoclastic differentiation of MBMM???ncells (MCSF dependent bone marrow derived macrophage) were studied. The osteoclastic differentiation was induced with MCSF (Macrophage colony-stimulating factor) and RANKL (receptor for activation of nuclear factor kappa B ligand). The number of multinucleated and TRAP-positive osteoclasts (n≧3) and s-TRAP activity per well are assessed. Six out of the seven flavones studied were all found to inhibit osteoclastogenesis in a dose-dependent manner. On the other hand, the effects of 7 flavones on osteogenesis of MC3T3 cells (pre-osteoblast cell) were also assessed. The alkaline phosphates activities of the flavone treated MC3T3 cells in osteogenic media were determined. Only 6-hydroxyflavone and 7-mthoxy flavone consistently increased the formation of osteoblasts in a dose-dependent manner. While 5-mthoxyflavone、baicalein、luteolin inhibited the osteogenic medium-induced MC3T3 ALP activities. In addition , 7-methoxy flavones was also dose-dependently enhanced calcium deposition on MC3T3 cells in osteogenic media for 20 days.
In conclusion, among the 7 flavones screened, only 7-methoxy flavone and 6-hydroxy flavone were capable of inhibiting osteoclastogenesis of MBMM???ncells and simultaneously promoting bone formation of pre-osteoblasts. These two flavones have the potential to became parts of beneficial remedy for preventing from or therapeutic treatment for osteoporosis.
論文目次 目錄
致謝…i
目錄…ii
縮寫表 (Abbreviation)…v
圖索引…vii
中文摘要…1
Abstract…3
I. 緒論…5
一、 骨的構造…5
二、 骨骼的組成…6
三、 骨細胞…7
四、 造骨細胞….7
五、 蝕骨細胞….8
六、 骨的重塑作用….9
七、 骨質疏鬆症…10
II. 研究目的…14
III. 實驗材料及方法…15
一、 材料…15
二、 實驗方法….16
1. 小鼠MBMM?眵茩M之分離與培養…16
2. 小鼠MBMM?眵茩M分化實驗…16
3. 細胞毒性之測定(MTT assay)…17
4. 鹼性磷酸酶測定(Alkaline phosphatase,ALP)…17
5. 酸性磷酸脢染色( Tartrate-resistant acid phosphatase ,TRAP )…18
6. 酸性磷酸脢活性測試 (TRAP activity assay)…18
7. Alizarin Red S染色…18
8. SDS-PAGE與Western blot之操作…19
9. 統計方法…21
IV. 實驗結果分析…22
一. 黃酮類化合物對MBMM之分化為蝕骨細胞的影響…22
1. MBMM?眵茩M之分化與定性驗…22
2. 黃酮類化合物對MBMM的毒殺驗…22
3. 黃酮類化合物對MBMM分化的響…23
4. 7-Methoxy flavone對MBMM分化的響…24
二. 黃酮類化合物對RAW264.7細胞分化的影響…25
7-Methoxy flavone對RAW264.7細胞分化的影響…25
三. 黃酮類化合物對MC3T3細胞骨化的影響…25
1. 前造骨細胞(MC3T3)骨化實驗…25
2. 黃酮類化合物對前造骨細胞(MC3T3)毒性實驗…26
3. 黃酮類化合物對前造骨細胞(MC3T3)分化實驗…26
4. 7-methoxy flavone及6-hydroxy flavone對前造骨細胞
(MC3T3)骨化實驗…26

四. 7-methoxy flavone抑制RAW264.7細胞蝕骨化之可能訊息傳遞路徑…27
7-methoxy flavone 對RAW264.7細胞內ERK蛋白磷酸化的影響…27
V. 結論…28
VI. 討論…29
VII. 未來展望…33
VIII. 圖表…34
IX. 參考文獻…60
X. Appendex…69
參考文獻 IX. 參考文獻 (References)
1. Sims NA, Gooi JH (2008) Bone remodeling: Multiple cellular interactions required for coupling of bone formation and resorption. Seminars in Cell and Developmental Biology. 19:444-451

2. Keen R. (2007) Osteoporosis: strategies for prevention and management. Best Practice and Research Clinical Rheumatology. 21:109-122

3. Knothe Tate ML, Adamson JR, Tami AE, Bauer TW. (2004) The osteocyte. The International Journal of Biochemistry and Cell Biology. 36:1-8

4. Ducy P, Zhang R, Geoffroy V, Ridall A L, and Karsenty G. (1997) Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation. Cell. 89:747-754

5. Datta HK, Ng WF, Walker JA, Tuck SP and Varanasi SS. (2008) The cell biology of bone metabolism. Journal of Clinical Pathology. 61:577-587

6. Asou Y , Rittling SR, Yoshitake H , Tsuji K , Shinomiya K , Nifuji A , Denhardt DT, and Noda M . (2001) Osteopontin Facilitates Angiogenesis, Accumulation of Osteoclasts, and Resorption in Ectopic Bone. Endocrinology. 142:1325-1332

7. Malaval L, Wade-Guéye N M, Boudiffa M, Fei J, Zirngibl R, Chen F, Laroche N, Roux J P, Burt-Pichat B, Duboeuf F, Boivin G, Jurdic P, Lafage-Proust M H, Amédée J, Vico L, Rossant J and Aubin J E. (2008)
Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. The Journal of Experimental Medicine. 205:1145-1153

8. Ogita M, Rached M T , Dworakowski E, Bilezikian J. P., and Kousteni S. (2008) Differentiation and Proliferation of Periosteal Osteoblast Progenitors Are Differentially Regulated by Estrogens and Intermittent Parathyroid Hormone Administration. Endocrinology. 149:5713-5723

9. Wu X, Zeng LH, Taniguchi T and Xie QM. (2007) Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells. Cell Death and Differentiation. 14:1792-1801

10. Dufour C, Holy X, and Marie PJ. (2008) Transforming growth factor-???nprevents osteoblast apoptosis induced by skeletal unloading via PI3K/Akt, Bcl-2, and phospho-Bad signaling. Am J Physiol Endocrinol Metab. 294:794-801

11. Martin TJ and Sims NA . (2005) Osteoclast-derived activity in the coupling of bone formation to resorption. Molecular Medicine. 11:76-81

12. Boyle WJ, Simonet WS and Lacey DL. (2003) Osteoclast differentiation and activation. Nature. 423:337-342

13. Granholm S , Lundberg P and Lerner UH. (2007) Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-kB and c-Fms. Journal of Endocrinology. 195:415-427

14. Chiao JW, Moonga BS, Yang YM, Kancherla R, Mittelman A, Wu-Wong JR and Ahmed T. (2000) Endothelin-1 from prostate cancer cells is enhanced by bone contact which blocks osteoclastic bone resorption. British Journal of Cancer. 83:360-365

15. Li X , Qin L , Bergenstock M , Bevelock LM, Novack DV, and Partridge NC. (2007) Parathyroid Hormone Stimulates Osteoblastic Expression of MCP-1 to Recruit and Increase the Fusion of Pre/Osteoclasts. THE Journal of Biological Chemistry. 282:33098-33106

16. Palacios VG, Robinson LJ, Borysenko CW, Lehmann T, Kalla SE, and Blair HC. (2005) Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens. The Journal of Biological Chemistry. 280:13720-13727

17. Giuliani N, Colla S, Morandi F, Rizzoli V. (2004)The RANK/RANK ligand system is involved in interleukin-6 and interleukin-11 up-regulation by human myeloma cells in the bone marrow microenvironment. Haematologica. 89:1118-23

18. Sanuki R, Shionome C, Kuwabara A, Mitsui N, Koyama Y, Suzuki N, Zhang F, Shimizu N, Maeno M.(2010) Compressive force induces osteoclast differentiation via prostaglandin E2 production in MC3T3-E1 cells. Connective tissue research. 51:150-8

19. Lewiecki EM , MD. (2009) Managing osteoporosis:Challenges and strategies. Cleveland Clinic Journal of Medicine. 76:457-466

20. Tinetti ME, Speechley M. (1989) Prevention of falls among the elderly. New England Journal of Medicine. 320:1055–1059.

21. Winner SJ, Morgan CA, Evans JG. (1989) Perimenopausal risk of falling and incidence of distal forearm fracture. British Medical ournal. 298:1486-1488.

22. Orwoll ES et al. (1996) Axial bone mass in older women. Study of Osteoporotic Fractures Research Group. Annals of Internal Medicine. 124:187-196.

23. Johnston CC, Slemenda CW. (1998)Pathogenesis of postmenopausal osteoporotic fractures. Osteoporosis. 53-64.

24. Melton LJ et al. (1999) Vertebral fractures predict subsequent fractures.Osteoporosis International. 10:214-221.

25. Seeman E. (1996) The effects of tobacco and alcohol use on bone. In: Marcus R, Feldman D, Kelsey J, eds. Osteoporosis. San Diego, CA, Academic Press. 577-597.

26. Naves-Diaz M, O’Neill TW, Silman A. (1997) The influence of alcohol consumption on the risk of vertebral deformity. The European Vertebral Osteoporosis Study Group. Osteoporosis International. 7:65-71.

27. Marcus R. (1996) Mechanisms of exercise effects on bone. In: Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of bone biology. San Diego, CA Academic Press. 1135-1146.

28. Ralston SH (2010) Genetics of osteoporosis. Annals of the New York Academy of Sciences. 181-189

29. Kanis JA. (1997) Osteoporosis. Oxford, Blackwell Science.

30. Dawson-Hughes B, Dallal GE, Krall EA, Sadowski L, Sahyoun N, Tannenbaum S . (1990) A controlled trial of the effect of calcium supplementation bone density in postmenopausal women. The New England Journal of Medicine 323: 878-883.

31. 31Jackson RD, LaCroix AZ, Gass M et al. (2006) Calcium plus vitamin D supplementation and the risk of fractures. The New England Journal of Medicine. 354:669-683.

32. Hock JM and Gera I. (1992) Effects of continuous and intermittent administration and inhibition of resorption on the anabolic response of bone to parathyroid hormone. Journal of Bone and Mineral Research. 7:65-72

33. Russell RGG and Rogers M. (1999) Bisphosphonates: from the laboratory to the clinic and back again. Bone. 25:97–106.

34. Girasole G, Jilka RL, Passeri G, Boswell S, Boder G, Williams DC and Manolagas SC. (1992) 17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro:a potential mechanism for the antiosteoporotic effect of estrogens. The Journal of Clinical Investigation. 89:883-91.

35. Ross J A and Kasum C M. (2002) Dietary Flavonoid:Bioavailability, Metabolic Effects, and Safety. Annual Review of Nutrition. 22:19–34

36. Barnes S (2010) The biochemistry, chemistry and physiology of the isoflavones in soybeans and their food products. Lymphatic research and biology. 1:89-98

37. Serafini M, Peluso I, Raguzzini A(2010) Flavonoids as anti-inflammatory agents. The Proceedings of the Nutrition Society. 69:273-278

38. 38 Han JJ, Hao J, Kim CH, Hong JS, Ahn HY, Lee YS(2009) Quercetin prevents cardiac hypertrophy induced by pressure overload in rats. Journal of Veterinary Medical Science. 71:737-43

39. Middleton E Jr, Kandaswami C, and Theoharides T C. (2000) The Effects of Plant Flavonoids on Mammalian Cells: Implications for Inflammation, Heart Disease, and Cancer. Pharmacological Reviews. 52:673–751

40. Jeon BJ, Ahn J, Kwak HS (2009) Effect of isoflavone-enriched milk on bone mass in ovariectomized rats. Journal of Medicinal Food. 12:1260-1267

41. Qu L, Chen H, Liu X, Bi L, Xiong J, Mao Z, Li Y. (2010)
Protective Effects of Flavonoids Against Oxidative Stress Induced by Simulated Microgravity in SH-SY5Y Cells. Neurochemical research

42. Chen C Y, Peng W H, Tsai K D, Hsu S L (2007) Luteolin suppresses inflammation-associated gene expression by blocking NF-κB and AP-1 activation pathway in mouse alveolar macrophages. Life Sciences. 81 :1602– 1614


43. Chiu F L and Lin J K. (2008) Downregulation of Androgen Receptor Expression by Luteolin Causes Inhibition of Cell Proliferation and Induction of Apoptosis in Human Prostate Cancer Cells and Xenografts. The Prostate. 68:61-71

44. Zhang J, Ahn MJ, Sun QS, Kim KY, Hwang YH, Ryu JM, Kim J. (2008) Inhibitors of bone resorption from Halenia corniculata. Archives of pharmacal research. 31:850-855

45. Kim MH, Ryu SY, Bae MA, Choi JS, Min YK, Kim SH. (2008) Baicalein inhibits osteoclast differentiation and induces mature osteoclast apoptosis. Food and Chemical Toxicology. 46:3375–3382

46. Choi EM (2007) Modulatory effects of luteolin on osteoblastic function andinflammatory mediators in osteoblastic MC3T3-E1 cells. Cell Biology International. 31:870-877

47. Sliwiński L, Folwarczna J, Janiec W, Grynkiewicz G, Kuzyk K (2005) Differential effects of genistein, estradiol and raloxifene on rat osteoclasts in vitro. Pharmacological reports. 57:352-359

48. Chen X, Garner SC, Quarles LD, Anderson JJ (2003) Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation. The Journal of nutritional biochemistry. 14:342-349

49. Wattel A, Kamel S, Mentaverri R, Lorget F, Prouillet C, Petit JP, Fardelonne P, Brazier M. (2003) Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochemical pharmacology. 65:35-42

50. Prouillet C, Mazière JC, Mazière C, Wattel A, Brazier M, Kamel S (2004) Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochemical pharmacology. 67:1307-1313

51. Kyung TW, Lee JE, Shin HH, Choi HS (2008) Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB. Experimental & molecular medicine. 40:52-58

52. Horcajada MN, Habauzit V, Trzeciakiewicz A, Morand C, Gil-Izquierdo A, Mardon J, Lebecque P, Davicco MJ, Chee WS, Coxam V, Offord E (2008) Hesperidin inhibits ovariectomized-induced osteopenia and shows differential effects on bone mass and strength in young and adult intact rats. Journal of applied physiology. 104:648-54

------------------------------------------------------------------------ 第 14 筆 ---------------------------------------------------------------------
系統識別號 U0007-0402201010594000
論文名稱(中文) 以雙磷酸鹽類-螢光金奈米團簇早期偵測前列腺癌的骨轉移
論文名稱(英文) Eearly detection of bone metastasis of prostate cancer using bisphosphonate fluorescence gold nanocluster
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 1
出版年 99
研究生(中文) 陳蓓萱
學號 M121096009
學位類別 碩士
語文別 中文
口試日期 2010-01-13
論文頁數 45頁
口試委員 委員-蔡坤志
指導教授-賴文福
委員-施純明
關鍵字(中) 前列腺癌
分子影像
螢光金奈米團簇
關鍵字(英) prostate cancer
molecular imaging
nanogold cluster
學科別分類
中文摘要 目前在台灣男性十大死因中,癌症排名第一,其中前列腺癌,在癌症死亡率排名第七名,前列腺癌的初期沒有症狀,臨床上的診斷採用MRI、CT、X-ray、PET、SPECT等作為診斷工具,當被診斷出確定為前列腺癌時通常為末期或是癌細胞已經轉移到其他組織,這是造成前列腺癌死亡的主因,根據過去的統計發現前列腺癌經常轉移到骨頭與淋巴結,因為癌症細胞的轉移會導致生命危險,所以臨床上希望找到一個能在前列腺癌轉移初期就偵測到的方法。
本實驗研究的目的是合成出一個新的探針,利用光學分子影像的方式早期偵測前列腺癌的骨轉移。前列腺癌細胞轉移初期引發造骨細胞活化,使得骨密度增加,研究發現骨頭中的礦物質有70% 為氫氧基磷灰石(hydroxyapatite),因為成骨細胞受到前列腺癌細胞刺激骨細胞分泌大量氫氧基磷灰石行成新骨,因此利用氫氧基磷灰石來當作偵測前列腺癌骨早期轉移的標誌,過去的研究發現雙磷酸鹽類對氫氧基磷灰石具有高親和力,本實驗設計一個雙磷酸鹽類-螢光金奈米團簇製備探針(NanoAu –BpN),以偵測骨化作用。選擇螢光金奈米團簇是因為其奈米團簇具有發螢光的特性同時不易光漂白對於人體較不具毒性,此探針有利於之後在生物體上的應用。
首先將螢光金奈米團簇接枝上雙磷酸鹽類, EDC做為交聯劑,以合成NanoAu-BpN (NA-BpN) -HA 探針,接著測試HA與探針混合反應的專一性。由細胞實驗證實前列腺癌細胞(LNCaP)與成骨細胞(7F2)共培養後分泌鈣離子,NA-BpN 探針與細胞反應後由於探針具有發紅色螢光的特性,用近內紅外線影像系統偵測探針與細胞的影像。合成出來的NA-BpN探針確認是與HA有專一性的作用。因此利用此探針達到早期偵測前列腺癌骨轉移的效果,提供一種新的偵測前列腺癌骨轉移的方式。
英文摘要 Prostate cancer currently ranks the 2nd highest mortality rate in North America and 7th mortality rate in Taiwan. Prostate cancer frequently metastasizes to bone resulting in both osteoblastic and osteolytic lesions. Metastases from prostate cancer, most of which are adenocarinomas, nearly always form osteoblastic lesion in bone. The aim of my study is to early detect bone metastasis from prostate cancer. Bisphosphonate bind avidly to hydroxyapatite bone mineral surfaces. We synthesize a nanogold-bisphosphonate near-infrared fluorescence probes. Gold nanoparticles (nanoclusters) show the size-dependent fluorescence, no phtobleaching, broad excitation spectrum, and narrow emission spectrum. Gold nanoclusters (<2 nm) having near infrared fluorescence were utilized. Bisphosphates are bioconjugated to nanogold clusters by connecting agent EDC. The affinity between probes and hydroxyapatites was examined using molecular imaging system (IVIS-200). The molecular imaging probe of NanoAu-BpN can be specifically targeted to the prostate cancer cell-after 9 days ‘ co-cultivation of the prostate cancer cells osteoblasts induced bone formation. The NanoAu-BpN probe provides a new method to detect the bone metastasis of prostate cancer.
論文目次 章節目錄
中文摘要
英文摘要
第一章 緒論
一. 前列腺癌概論:
前列腺型態與特性 ………………………………………………….1
前列腺癌流行病學 ………………………………………………….1
前列腺癌病理學 …………………………………………………..…2
骨轉移 ..……………………………………………………………………2
癌症分期 ..……………………………………………………………..4
診斷前列腺癌方法……………………………………………………4
前列腺癌治療方法……………………………………………………6
二. 分子影像:…..………………………………………..………………….…..7
電腦電層掃描……………………………………………………….…..8
磁振造影.……..………………………..……..………..………….…….8
核子造影 …………………….……………….…………….…….……..9
光學影像 …………………………………….…….………….….……..9
超音波影像 …..………………………………………………….……..10
奈米螢光材料…………………………………………………….……..11
三. 雙磷酸鹽類……………………………………………………………………..14
第二章 實驗目的…..………………………………………………………………...17
第三章 材料與方法
一. 材料、試劑 ……………………………………………………………………18
二. 儀器設備………………………………………………………………………….19
三. 方法…….…………………………………………………………………………...20
第四章 實驗設計 ...…………………………………………………………………..25
第五章 實驗結果....……………………………………………………………………26
第六章 討論 ……………………………………………………….…………………..28
參考文獻 ………………………………………………………………………………..34
圖表 ………………………………………………………………………………………….37
附錄 ………………………………………………………………………………………….45
參考文獻 參考文獻
1. 梅約醫學中心. Mayo Clinic on Prostate Health-攝護腺. (2004).
2. McNeal, J.E. Normal histology of the prostate. Am J Surg Pathol 12, 619-33 (1988).
3. McNeal, J.E. Normal anatomy of the prostate and changes in benign prostatic hypertrophy and carcinoma. Semin Ultrasound CT MR 9, 329-34 (1988).
4. Miller Dc Fau - Hafez, K.S., Hafez Ks Fau - Stewart, A., Stewart A Fau - Montie, J.E., Montie Je Fau - Wei, J.T. & Wei, J.T. Prostate carcinoma presentation, diagnosis, and staging: an update form the National Cancer Data Base.
5. D., W. Radiology of bone tumors and allied disorders (Saunders, Philadelphia, 1982).
6. 張明澄. 攝護腺寶典. (1994).
7. Buijs, J.T. & van der Pluijm, G. Osteotropic cancers: from primary tumor to bone. Cancer Lett 273, 177-93 (2009).
8. Logothetis, C.J. & Lin, S.H. Osteoblasts in prostate cancer metastasis to bone. Nat Rev Cancer 5, 21-8 (2005).
9. 張延驊, 陳., 張心堤. 攝護腺瘤. (1995).
10. Fizazi, K. et al. Prostate cancer cells-osteoblast interaction shifts expression of growth/survival-related genes in prostate cancer and reduces expression of osteoprotegerin in osteoblasts. Clinical Cancer Research 9, 2587-2597 (2003).
11. Lin, D.L. et al. Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro. Prostate 47, 212-21 (2001).
12. Rentsch, C.A., Cecchini, M.G. & Thalmann, G.N. Loss of inhibition over master pathways of bone mass regulation results in osteosclerotic bone metastases in prostate cancer. Swiss Med Wkly 139, 220-5 (2009).
13. 江漢聲. 攝護腺-疾病與保健 (健康世界雜誌社, 台北市, 1983).
14. Weissleder, R. & Mahmood, U. Molecular imaging. Radiology 219, 316-33 (2001).
15. Rudin, M. Molecular Imaging (2005).
16. Paulus, M.J., Gleason, S.S., Easterly, M.E. & Foltz, C.J. A review of high-resolution X-ray computed tomography and other imaging modalities for small animal research. Lab Anim (NY) 30, 36-45 (2001).
17. Ritman, E.L. Molecular imaging in small animals--roles for micro-CT. J Cell Biochem Suppl 39, 116-24 (2002).
18. Sharma, P., Brown, S., Walter, G., Santra, S. & Moudgil, B. Nanoparticles for bioimaging. Adv Colloid Interface Sci 123-126, 471-85 (2006).
19. Gambhir, S.S. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2, 683-93 (2002).
20. Golden, J.P. & Ligler, F.S. A comparison of imaging methods for use in an array biosensor. Biosens Bioelectron 17, 719-25 (2002).
21. Frangioni, J.V. In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol 7, 626-34 (2003).
22. Hamaoka, T., Madewell, J.E., Podoloff, D.A., Hortobagyi, G.N. & Ueno, N.T. Bone imaging in metastatic breast cancer. J Clin Oncol 22, 2942-53 (2004).
23. Zheng, J., Zhang, C. & Dickson, R.M. Highly fluorescent, water-soluble, size-tunable gold quantum dots. Physical Review Letters 93 (2004).
24. Zaheer, A. et al. In vivo near-infrared fluorescence imaging of osteoblastic activity. Nature Biotechnology 19, 1148-1154 (2001).
25. Peng, Z.A. & Peng, X. Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor [6]. Journal of the American Chemical Society 123, 183-184 (2001).
26. Lin, C.A.J. et al. Design of an amphiphilic polymer for nanoparticle coating and functionalization. Small 4, 334-341 (2008).
27. Hansen, P.M., Bhatia, V.K., Harrit, N. & Oddershede, L. Expanding the optical trapping range of gold nanoparticles. Nano Lett 5, 1937-42 (2005).
28. Takagahara, T. & Takeda, K. Theory of the quantum confinement effect on excitons in quantum dots of indirect-gap materials. Phys Rev B Condens Matter 46, 15578-15581 (1992).
29. Alivisatos, A.P. Semiconductor clusters, nanocrystals, and quantum dots. Science 271, 933-937 (1996).
30. Yu, W.W. Semiconductor quantum dots: synthesis and water-solubilization for biomedical applications. Expert Opin Biol Ther 8, 1571-81 (2008).
31. Burton, J.B. et al. Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer. Nature Medicine 14, 882-888 (2008).
32. Hricak, H., Choyke, P.L., Eberhardt, S.C., Leibel, S.A. & Scardino, P.T. Imaging prostate cancer: A multidisciplinary perspective. Radiology 243, 28-53 (2007).
33. Larson, S.M. & Scho?der, H. Advances in positron emission tomography applications for urologic cancers. Current Opinion in Urology 18, 65-70 (2008).
34. Brown, J.E., Neville-Webbe, H. & Coleman, R.E. The role of bisphosphonates in breast and prostate cancers. Endocr Relat Cancer 11, 207-24 (2004).
35. Licata, A.A. Discovery, clinical development, and therapeutic uses of bisphosphonates. Ann Pharmacother 39, 668-77 (2005).
36. Viereck, V. et al. Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts. Biochem Biophys Res Commun 291, 680-6 (2002).
37. Lin, J.H. Bisphosphonates: a review of their pharmacokinetic properties. Bone 18, 75-85 (1996).
38. Koeneman, K.S., Yeung, F. & Chung, L.W. Osteomimetic properties of prostate cancer cells: a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment. Prostate 39, 246-61 (1999).
39. Mundy, G.R. Mechanisms of bone metastasis. Cancer 80, 1546-56 (1997).
40. Shah, B., Clark, P., Stroscio, M. & Mao, J. Labeling and imaging of human mesenchymal stem cells with quantum dot bioconjugates during proliferation and osteogenic differentiation in long term. Conf Proc IEEE Eng Med Biol Soc 1, 1470-3 (2006).
41. Nie, S., Xing, Y., Kim, G.J. & Simons, J.W. Nanotechnology applications in cancer. Annu Rev Biomed Eng 9, 257-88 (2007).
42. Biju, V., Itoh, T., Anas, A., Sujith, A. & Ishikawa, M. Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. Anal Bioanal Chem 391, 2469-95 (2008).
43. Zaheer, A. et al. Optical imaging of hydroxyapatite in the calcified vasculature of transgenic animals. Arterioscler Thromb Vasc Biol 26, 1132-6 (2006).
44. Fizazi, K. et al. Prostate cancer cells-osteoblast interaction shifts expression of growth/survival-related genes in prostate cancer and reduces expression of osteoprotegerin in osteoblasts. Clin Cancer Res 9, 2587-97 (2003).
45. Singh, A.S. & Figg, W.D. In vivo models of prostate cancer metastasis to bone. J Urol 174, 820-6 (2005).



------------------------------------------------------------------------ 第 15 筆 ---------------------------------------------------------------------
系統識別號 U0007-0408201015434800
論文名稱(中文) Hsp90抑制劑誘導HER2低表現乳癌細胞株細胞死亡的分子機制之探討
論文名稱(英文) Molecular mechanism of Hsp90 inhibitor-induced cell death in low HER2-expressing breast cancer cell line
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 謝美君
學號 M120097004
學位類別 碩士
語文別 中文
口試日期 2010-07-13
論文頁數 74頁
口試委員 指導教授-劉俊仁
委員-曾淑芬
委員-陳俊憲
關鍵字(中) Hsp90抑制劑
乳癌
關鍵字(英) Hsp90 inhibitor
breast cancer
學科別分類
中文摘要 熱休克蛋白90(Hsp90)為細胞中幫助蛋白折疊的一個重要蛋白質,因其特殊及重要的功能,近年來已成為抗癌藥物設計的標靶。由於造成乳癌轉移及惡性化的蛋白HER2(Human epiderminal growth factor receptor 2; ErbB2)也為Hsp90所調控的蛋白之一,因此獨立使用Hsp90抑制劑17-AAG (17-allyl-amino-demethoxy-geldanamycin)或與其他化療藥物合併使用,對高度表現HER2的乳癌細胞或對HER2高表現且Trastuzumab具抗藥性的乳癌細胞具很強的毒殺效果。然而並非所有的乳癌細胞皆為HER2過度表現,因此本研究目的在探討Hsp90抑制劑17-DMAG對於HER2低表現量的乳癌細胞株MCF7的細胞毒殺效果。藉由細胞毒性測試(sulforhodamine B, SRB assay)結果可知,17-DMAG (17-dimethylamino-ethylamino-17-demethoxygeldanamycin)在對MCF7/WT與具抗藥性MCF7/ADR細胞作用72小時後,其IC50之劑量為20 nM與1300 nM,由螢光免疫染色與細胞凋亡分析結果可看出MCF/WT經17-DMAG 400 nM作用後,細胞凋亡情形增加,但MCF7/ADR經17-DMAG 400 nM作用則無明顯細胞凋亡表現。從西方墨點法結果,17-DMAG作用可促進MCF7/WT內質網壓力相關蛋白GRP78表現,故推測17-DMAG造成的MCF7/WT細胞死亡可能是透過內質網壓力;隨17-DMAG作用時間增加,GRP78所促進的下游路徑相關蛋白active form ATF6、caspase-12以及ATF4表現量皆增加;具抗藥性MCF7/ADR細胞GRP78表現量較MCF7/WT細胞高,經17-DMAG 400 nM作用,內質網壓力相關蛋白無明顯增加。壓力反應會增加細胞對Hsp90抑制劑的抗藥性,而抑制壓力反應蛋白質如Hsp70及Hsp27可避免對Hsp90抑制劑產生抗藥性,本研究藉由抑制具抗藥性MCF7/ADR之GRP78表現量,發現經17-DMAG 作用72小時IC50劑量降低為60 nM,細胞凋亡增加,西方墨點法結果可知內質網壓力相關蛋白表現量ATF6、caspase12與ATF4皆較MCF7/ADR增加,故抑制抗藥性MCF7/ADR細胞株之GRP78表現量可增加細胞對17-DMAG之藥物敏感性與由17-DMAG引發的內質網壓力,促進細胞凋亡。由以上結果可推測17-DMAG作用於HER2低表現乳癌細胞株MCF7會促進內質網壓力而導致細胞死亡。
英文摘要 Heat shock protein 90 (Hsp90), which facilitate proteins folding, is now a target for anti-cancer drugs development. Because epidermal growth factor receptor 2 (HER2) that induces metastasis and malignance of breast cancer is one of Hsp90’s client proteins, usage of Hsp90 inhibitors was now being proofed to beneficial for breast cancer treatment, which with HER2-overexpression and/or Trastuzumab-resistnat. However, cytotoxic mechanisms of Hsp90 inhibitors for those breast cancer cells with low HER2 expression are unclear. Therefore, the molecular mechanism underlay 17-DMAG (17-dimethylamino-ethylamino-17-demethoxygeldanamycin) on low HER2 expression breast cancer cell line MCF7 was explored in this study. From the in vitro cytotoxicity test evaluated by NCI standard protocol (sulforhodamine B, SRB) showed that the IC50 of 17-DMAG in MCF7/WT and MCF7/ADR (drug resistant cell line) are 20 nM and 1300 nM. With time- dependent manner, treatment of MCF7/WT with 17-DMAG 400 nM could increase the population of cells with DNA condensation observed under microscope and cell apoptosis by flow cytometry. It indicated that 17-DMAG treatment could lead to cell apoptosis. However, 17-DMAG-induced apoptosis was not observed in MCF7/ADR. Further examination by western blot analysis showed that the level of ER stress chaperone protein GRP78 increased in time-dependent manner in MCF7/WT, but not MCF7/ADR. It suggested that MCF7/WT cell death-induced by 17-DMAG might through drug-induced ER stress pathway. Further analysis showed that cellular levels of cleavaged-ATF6, caspase 12 and ATF4, down-stream pathway of GRP78, increasing with time-dependent manner in MCF7/WT, but not MCF7/ADR. Otherwise, knockdown GRP78 expression in MCF7/ADR could increase drug sensitivity ( 72h IC50=60 nM ) and the expression of ER stress related proteins. From all the data collected, the molecule mechanism of cell death induced by 17-DMAG in low HER2-expression MCF7 cell line was through drug-induced ER stress pathway.
論文目次 目錄
致 謝……………………………………………II
中文摘要……………………………………………III
英文摘要 ……………………………………………IV
縮寫表………………………………………………V
一、背景
1.1 熱休克蛋白 (heat shock proteins;HSPs)………… 1
1.2 HSP90 (Heat Shock Protein 90)……………………… 2
1.3 乳癌…………………………………………………………6
1.4 Hsp90 抑制劑………………………………………………8
1.5 Hsp90抑制劑在乳癌方面的作用…………………………11
1.6 Hsp90抑制劑與抗藥性之關係……………………………12
二、研究特定目標 ……………………………………………14
三、材料與方法(包括實驗方法及進行步驟)
3.1實驗細株……………………………………………………15
3.2 細胞培養…… ……………………………………………15
3.3藥物對細胞毒性測試(sulforhodamine B, SRB assay) …16
3.4免疫細胞染色( Immunocytochemistry ) …………………17
3.5流式細胞儀分析細胞凋亡 (Annexin V-PI costain) ……18
3.6蛋白質表現定…………………………………………………19
四、結果…………………………………………………………27
五、討論…………………………………………………………33
六、參考資料……………………………………………………40
七、圖表…………………………………………………………55

參考文獻 1. Chen X, Easton D, Oh HJ, Lee-Yoon DS, Liu X, Subjeck J. The 170 kDa glucose regulated stress protein is a large HSP70-, HSP110-like protein of the endoplasmic reticulum. FEBS Lett. 1996 Feb 12;380(1-2):68-72.

2. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 1992 Oct;72(4):1063-81.

3. Kregel KC. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol. 2002 May;92(5):2177-86.

4. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 1998 Dec 15;12(24):3788-96.

5. Sharma SK, Christen P, Goloubinoff P. Disaggregating chaperones: an unfolding story. Curr Protein Pept Sci. 2009 Oct;10(5):432-46.

6. Feder ME, Hofmann GE. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol. 1999;61:243-82.

7. Moseley P. Stress proteins and the immune response. Immunopharmacology. 2000 Jul 25;48(3):299-302.

8. Becker J, Craig EA. Heat-shock proteins as molecular chaperones. Eur J Biochem. 1994 Jan 15;219(1-2):11-23.

9. Pakravan N, Langroudi L, Hajimoradi M, Hassan ZM.Co-administration of GP96 and Her2/neu DNA vaccine in a Her2 breast cancer model. Cell Stress Chaperones. 2010 Jun 12.

10. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002 Mar 8;295(5561):1852-8.

11. Tavaria M, Gabriele T, Kola I, Anderson RL. A hitchhiker's guide to the human Hsp70 family. Cell Stress Chaperones. 1996 Apr;1(1):23-8.

12. Whitham M, Fortes MB. Heat shock protein 72: release and biological significance during exercise. Front Biosci. 2008 Jan 1;13:1328-39.

13. Wong HR, Menendez IY, Ryan MA, Denenberg AG, Wispé JR. Increased expression of heat shock protein-70 protects A549 cells against hyperoxia. Am J Physiol. 1998 Oct;275(4 Pt 1):L836-41.

14. Sarto C, Binz PA, Mocarelli P. Heat shock proteins in human cancer. Electrophoresis. 2000 Apr;21(6):1218-26.

15. Grenert JP, Sullivan WP, Fadden P, Haystead TA, Clark J, Mimnaugh E, Krutzsch H, Ochel HJ, Schulte TW, Sausville E, Neckers LM, Toft DO.The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation. J Biol Chem. 1997 Sep 19;272(38):23843-50.

16. Isaacs JS, Xu W, Neckers L. Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell. 2003 Mar;3(3):213-7.

17. Pratt WB, Toft DO. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood). 2003 Feb;228(2):111-33.

18. Jennifer R. Smith, Paul Workman. Targeting the cancer chaperone HSP90. Drug Discovery Today: Therapeutic Strategies.2007;4(4):219-27.

19. Goetz CA, O'Neil JJ, Farrar MA. Membrane localization, oligomerization, and phosphorylation are required for optimal raf activation. J Biol Chem. 2003 Dec 19;278(51):51184-9.

20. Kamal A, Boehm MF, Burrows FJ. Therapeutic and diagnostic implications of Hsp90 activation. Trends Mol Med. 2004 Jun;10(6):283-90.

21. Maloney A, Workman P. HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther. 2002 Jan;2(1):3-24.

22. Sreedhar AS, Kalmár E, Csermely P, Shen YF. Hsp90 isoforms: functions, expression and clinical importance. FEBS Lett. 2004 Mar 26;562(1-3):11-5.

23. Nimmanapalli R, O'Bryan E, Bhalla K. Geldanamycin and its analogue 17-allylamino-17-demethoxygeldanamycin lowers Bcr-Abl levels and induces apoptosis and differentiation of Bcr-Abl-positive human leukemic blasts. Cancer Res. 2001 Mar 1;61(5):1799-804.

24. Nagata Y, Anan T, Yoshida T, Mizukami T, Taya Y, Fujiwara T, Kato H, Saya H, Nakao M. The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association. Oncogene. 1999 Oct 28;18(44):6037-49.

25. Blagosklonny MV, Toretsky J, Bohen S, Neckers L. Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8379-83.

26. Yang S, Qu S, Perez-Tores M, Sawai A, Rosen N, Solit DB, Arteaga CL. Association with HSP90 inhibits Cbl-mediated down-regulation of mutant epidermal growth factor receptors. Cancer Res. 2006 Jul 15;66(14):6990-7.

27. Beck R, Verrax J, Gonze T, Zappone M, Pedrosa RC, Taper H, Feron O, Calderon PB. Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death. Biochem Pharmacol. 2009 Feb 1;77(3):375-83.

28. Schröder M, Kaufman RJ. The mammalian unfolded protein response. Annu Rev Biochem. 2005;74:739-89.

29. Lee AS. GRP78 induction in cancer: therapeutic and prognostic implications. Cancer Res. 2007 Apr 15;67(8):3496-9.

30. Kaufman RJ.Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 1999 May 15;13(10):1211-33.

31. Lee AS. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci. 2001 Aug;26(8):504-10.

32. Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004 Apr;11(4):381-9.

33. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell. 2003 Mar;11(3):619-33.

34. Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcriptional and translational control in the Mammalian unfolded protein response. Annu Rev Cell Dev Biol. 2002;18:575-99..

35. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000 Jun;2(6):326-32.

36. Hengstermann A, Müller T. Endoplasmic reticulum stress induced by unfolded-protein-response-dependent PERK pathway of cell survival. Free Radic Biol Med. 2008 Mar 15;44(6):1097-107.

37. Wouters BG, van den Beucken T, Magagnin MG, Koritzinsky M, Fels D, Koumenis C. Control of the hypoxic response through regulation of mRNA translation. Semin Cell Dev Biol. 2005 Aug-Oct;16(4-5):487-501.

38. Inagi R. Endoplasmic reticulum stress in the kidney as a novel mediator of kidney injury. Nephron Exp Nephrol. 2009;112(1):e1-9.

39. Chami M, Oulès B, Szabadkai G, Tacine R, Rizzuto R, Paterlini-Bréchot P. Role of SERCA1 truncated isoform in the proapoptotic calcium transfer from ER to mitochondria during ER stress. Mol Cell. 2008 Dec 5;32(5):641-51.

40. Sano R, Annunziata I, Patterson A, Moshiach S, Gomero E, Opferman J, Forte M, d'Azzo A. GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis. Mol Cell. 2009 Nov 13;36(3):500-11.

41.行政院衛生署 統計資料
http://www.doh.gov.tw/CHT2006/DM/DM2_2.aspx?now_fod_list_no=10238&class_no=440&level_no=1

42.Yuan S, Shi C, Liu L, Han W. MUC1-based recombinant Bacillus Calmette-Guerin vaccines as candidates for breast cancer immunotherapy. Expert Opin Biol Ther. 2010 Jul;10(7):1037-48

43. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lønning P, Børresen-Dale AL. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10869-74.

44. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007 Aug 1;13(15 Pt 1):4429-34.

45. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lønning PE, Børresen-Dale AL, Brown PO, Botstein D. Molecular portraits of human breast tumours. Nature. 2000 Aug 17;406(6797):747-52.

46. Hu Z, Fan C, Oh DS, Marron JS, He X, Qaqish BF, Livasy C, Carey LA, Reynolds E, Dressler L, Nobel A, Parker J, Ewend MG, Sawyer LR, Wu J, Liu Y, Nanda R, Tretiakova M, Ruiz Orrico A, Dreher D, Palazzo JP, Perreard L, Nelson E, Mone M, Hansen H, Mullins M, Quackenbush JF, Ellis MJ, Olopade OI, Bernard PS, Perou CM. The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genomics. 2006 Apr 27;7:96.

47. Burstein HJ, Kuter I, Campos SM, Gelman RS, Tribou L, Parker LM, Manola J, Younger J, Matulonis U, Bunnell CA, Partridge AH, Richardson PG, Clarke K, Shulman LN, Winer EP. Clinical activity of trastuzumab and vinorelbine in women with HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2001 May 15;19(10):2722-30.

48. Tagliabue E, Balsari A, Campiglio M, Pupa SM. HER2 as a target for breast cancer therapy. Expert Opin Biol Ther. 2010 May;10(5):711-24.

49. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989 May 12;244(4905):707-12.

50. Spector NL, Xia W, Burris H 3rd, Hurwitz H, Dees EC, Dowlati A, O'Neil B, Overmoyer B, Marcom PK, Blackwell KL, Smith DA, Koch KM, Stead A, Mangum S, Ellis MJ, Liu L, Man AK, Bremer TM, Harris J, Bacus S. Study of the biologic effects of lapatinib, a reversible inhibitor of ErbB1 and ErbB2 tyrosine kinases, on tumor growth and survival pathways in patients with advanced malignancies. J Clin Oncol. 2005 Apr 10;23(11):2502-12.

51. Agus DB, Akita RW, Fox WD, Lewis GD, Higgins B, Pisacane PI, Lofgren JA, Tindell C, Evans DP, Maiese K, Scher HI, Sliwkowski MX. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell. 2002 Aug;2(2):127-37.

52. Di Cosimo S, Baselga J. Management of breast cancer with targeted agents: importance of heterogeneity. Nat Rev Clin Oncol. 2010 Mar;7(3):139-47.

53. Dancey J, Sausville EA. Issues and progress with protein kinase inhibitors for cancer treatment. Nat Rev Drug Discov. 2003 Apr;2(4):296-313.

54. Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, Petersen R, Frost P, Gibbons JJ, Wu H, Sawyers CL. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10314-9.

55.Giménez Ortiz A, Montalar Salcedo J. Heat shock proteins as targets in oncology. Clin Transl Oncol. 2010 Mar;12(3):166-73.

56. Modi S, Stopeck AT, Gordon MS, Mendelson D, Solit DB, Bagatell R, Ma W, Wheler J, Rosen N, Norton L, Cropp GF, Johnson RG, Hannah AL, Hudis CA. Combination of trastuzumab and tanespimycin (17-AAG, KOS-953) is safe and active in trastuzumab-refractory HER-2 overexpressing breast cancer: a phase I dose-escalation study. J Clin Oncol. 2007 Dec 1;25(34):5410-7.

57. Meyer P, Prodromou C, Hu B, Vaughan C, Roe SM, Panaretou B, Piper PW, Pearl LH. Structural and functional analysis of the middle segment of hsp90: implications for ATP hydrolysis and client protein and cochaperone interactions. Mol Cell. 2003 Mar;11(3):647-58.

58. Garnier C, Lafitte D, Tsvetkov PO, Barbier P, Leclerc-Devin J, Millot JM, Briand C, Makarov AA, Catelli MG, Peyrot V.Binding of ATP to heat shock protein 90: evidence for an ATP-binding site in the C-terminal domain. J Biol Chem. 2002 Apr 5;277(14):12208-14.

67. Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, Burrows FJ. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature. 2003 Sep 25;425(6956):407-10.

68. Jennifer R. Smith, Paul Workman. Targeting the cancer chaperone HSP90.
Cancer. 2007 Winter; 219-227

69. Supko JG, Hickman RL, Grever MR, Malspeis L. Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent. Cancer Chemother Pharmacol. 1995;36(4):305-15.

70. Soga S, Shiotsu Y, Akinaga S, Sharma SV. Development of radicicol analogues. Curr Cancer Drug Targets. 2003 Oct;3(5):359-69.

71. Wang M, Shen G, Blagg BS. Radanamycin, a macrocyclic chimera of radicicol and geldanamycin. Bioorg Med Chem Lett. 2006 May 1;16(9):2459-62. Epub 2006 Feb 7.

72.Shen G, Blagg BS. Radester, a novel inhibitor of the Hsp90 protein folding machinery. Org Lett. 2005 May 26;7(11):2157-60.

73. Schulte TW, Neckers LM. The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin. Cancer Chemother Pharmacol. 1998;42(4):273-9.

74. Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA. Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res. 2001 May 15;61(10):4003-9.

75. Ge J, Normant E, Porter JR, Ali JA, Dembski MS, Gao Y, Georges AT, Grenier L, Pak RH, Patterson J, Sydor JR, Tibbitts TT, Tong JK, Adams J, Palombella VJ. Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J Med Chem. 2006 Jul 27;49(15):4606-15.

76.Hollingshead M, Alley M, Burger AM, Borgel S, Pacula-Cox C, Fiebig HH, Sausville EA. In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol. 2005 Aug;56(2):115-25. Epub 2005 Mar 25.
77. Chiosis G. Discovery and development of purine-scaffold Hsp90 inhibitors. Curr Top Med Chem. 2006;6(11):1183-91.
78. Chiosis G, Lucas B, Shtil A, Huezo H, Rosen N. Development of a purine-scaffold novel class of Hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of Her2 tyrosine kinase. Bioorg Med Chem. 2002 Nov;10(11):3555-64.
79. Vilenchik M, Solit D, Basso A, Huezo H, Lucas B, He H, Rosen N, Spampinato C, Modrich P, Chiosis G. Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumor Hsp90. Chem Biol. 2004 Jun;11(6):787-97.
80. Lundgren K. et al. Abstract B161. Molecular Targets and Cancer Therapeutics AACR-NCI-EORTC International Conference. 2007 Abstract B161
81. Porter JR, Fritz CC, Depew KM. Discovery and development of Hsp90 inhibitors: a promising pathway for cancer therapy. Curr Opin Chem Biol. 2010 Jun;14(3):412-20. Epub 2010 Apr 19.
82. Sharp SY, Boxall K, Rowlands M, Prodromou C, Roe SM, Maloney A, Powers M, Clarke PA, Box G, Sanderson S, Patterson L, Matthews TP, Cheung KM, Ball K, Hayes A, Raynaud F, Marais R, Pearl L, Eccles S, Aherne W, McDonald E, Workman P. In vitro biological characterization of a novel, synthetic diaryl pyrazole resorcinol class of heat shock protein 90 inhibitors. Cancer Res. 2007 Mar 1;67(5):2206-16.
83. Sharp SY, Prodromou C, Boxall K, Powers MV, Holmes JL, Box G, Matthews TP, Cheung KM, Kalusa A, James K, Hayes A, Hardcastle A, Dymock B, Brough PA, Barril X, Cansfield JE, Wright L, Surgenor A, Foloppe N, Hubbard RE, Aherne W, Pearl L, Jones K, McDonald E, Raynaud F, Eccles S, Drysdale M, Workman P. Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues. Mol Cancer Ther. 2007 Apr;6(4):1198-211.
84. Smith V, Sausville E, Camlier R, Fiebig H, Burger A.17-DMAG (NSC 707545), a water-soluble geldanamycin analog, has superior in vitro and in vivo antitumor activity compared to the hsp90 inhibitor 17-AAG. Eur J Cancer 2002; 38(S7):S60.
85. Neckers L. Hsp90 inhibitors as novel cancer chemotherapeutic agents. Trends Mol Med. 2002;8(4 Suppl):S55-61.
86. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987 Jan 9;235(4785):177-82.
87. Leow CC, Chesebrough J, Coffman KT, Fazenbaker CA, Gooya J, Weng D, Coats S, Jackson D, Jallal B, Chang Y. Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib. Mol Cancer Ther. 2009 Aug;8(8):2131-41.
88. Modi S, Stopeck AT, Gordon MS, Mendelson D, Solit DB, Bagatell R, Ma W, Wheler J, Rosen N, Norton L, Cropp GF, Johnson RG, Hannah AL, Hudis CA. Combination of trastuzumab and tanespimycin (17-AAG, KOS-953) is safe and active in trastuzumab-refractory HER-2 overexpressing breast cancer: a phase I dose-escalation study. J Clin Oncol. 2007 Dec 1;25(34):5410-7.
89. Nguyen DM, Lorang D, Chen GA, Stewart JH 4th, Tabibi E, Schrump DS. Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis. Ann Thorac Surg. 2001 Aug;72(2):371-8; discussion 378-9.
90. Basso AD, Solit DB, Munster PN, Rosen N. Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2. Oncogene. 2002 Feb 14;21(8):1159-66.
91. Palacios C, López-Pérez AI, López-Rivas A. Down-regulation of RIP expression by 17-dimethylaminoethylamino-17-demethoxygeldanamycin promotes TRAIL-induced apoptosis in breast tumor cells. Cancer Lett. 2010 Jan 28;287(2):207-15.
92. Zajac M, Moneo MV, Carnero A, Benitez J, Martínez-Delgado B. Mitotic catastrophe cell death induced by heat shock protein 90 inhibitor in BRCA1-deficient breast cancer cell lines. Mol Cancer Ther. 2008 Aug;7(8):2358-66.
93. Roforth MM, Tan C. Combination of rapamycin and 17-allylamino-17-demethoxygeldanamycin abrogates Akt activation and potentiates mTOR blockade in breast cancer cells. Anti cancer Drugs. 2008 Aug;19(7):681-8.
94. Raja SM, Clubb RJ, Bhattacharyya M, Dimri M, Cheng H, Pan W, Ortega-Cava C, Lakku-Reddi A, Naramura M, Band V, Band H. A combination of Trastuzumab and 17-AAG induces enhanced ubiquitinylation and lysosomal pathway-dependent ErbB2 degradation and cytotoxicity in ErbB2-overexpressing breast cancer cells. Cancer Biol Ther. 2008 Oct;7(10):1630-40.
95. McCollum AK, TenEyck CJ, Stensgard B, Morlan BW, Ballman KV, Jenkins RB, Toft DO, Erlichman C. P-Glycoprotein-mediated resistance to Hsp90-directed therapy is eclipsed by the heat shock response. Cancer Res. 2008 Sep 15;68(18):7419-27.
96. Ueda K, Cardarelli C, Gottesman MM, Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A. 1987 May;84(9):3004-8.
97. Tsuruo T, Naito M, Tomida A, Fujita N, Mashima T, Sakamoto H, Haga N. Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal. Cancer Sci. 2003 Jan;94(1):15-21.
98. Kioka N, Yamano Y, Komano T, Ueda K. Heat-shock responsive elements in the induction of the multidrug resistance gene (MDR1). FEBS Lett. 1992 Apr 13;301(1):37-40
99. Guo F, Rocha K, Bali P, Pranpat M, Fiskus W, Boyapalle S, Kumaraswamy S, Balasis M, Greedy B, Armitage ES, Lawrence N, Bhalla K. Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin. Cancer Res. 2005 Nov 15;65(22):10536-44.
100. Schmitt E, Maingret L, Puig PE, Rerole AL, Ghiringhelli F, Hammann A, Solary E, Kroemer G, Garrido C. Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma. Cancer Res. 2006 Apr 15;66(8):4191-7.
101. Guo F, Rocha K, Bali P, Pranpat M, Fiskus W, Boyapalle S, Kumaraswamy S, Balasis M, Greedy B, Armitage ES, Lawrence N, Bhalla K. Abrogation of heat shock protein 70 induction as a strategy to increase antileukemia activity of heat shock protein 90 inhibitor 17-allylamino-demethoxy geldanamycin. Cancer Res. 2005 Nov 15;65(22):10536-44.
102. Schmitt E, Maingret L, Puig PE, Rerole AL, Ghiringhelli F, Hammann A, Solary E, Kroemer G, Garrido C. Heat shock protein 70 neutralization exerts potent antitumor effects in animal models of colon cancer and melanoma. Cancer Res. 2006 Apr 15;66(8):4191-7.
103. Cho HY, Thomas S, Golden EB, Gaffney KJ, Hofman FM, Chen TC, Louie SG, Petasis NA, Schönthal AH.(2009) Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress. Cancer Lett. 282(1):87-97
104. Niu G, Carter WB. Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways. Cancer Res. 2007 Feb 15;67(4):1487-93.
105. Smith-Jones PM, Solit DB, Akhurst T, Afroze F, Rosen N, Larson SM. Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors. Nat Biotechnol. 2004 Jun;22(6):701-6.
106. Di Cosimo S, Baselga J. Targeted therapies in breast cancer: where are we now? .Eur J Cancer. 2008 Dec;44(18):2781-90.
107. Böhmer RM, Morstyn G. Uptake of hematoporphyrin derivative by normal and malignant cells: effect of serum, pH, temperature, and cell size. Cancer Res. 1985 Nov;45(11 Pt 1):5328-34.
108. Taiyab A, Sreedhar AS, Rao ChM. Hsp90 inhibitors, GA and 17AAG, lead to ER stress-induced apoptosis in rat histiocytoma. Biochem Pharmacol. 2009 Jul 15;78(2):142-52.
109. Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol. 2000 Aug 21;150(4):887-94.
110. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4,participates in cytochrome c-dependent activation of caspase-3. Cell. 1997 Aug 8;90(3):405-13.
111. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001 Apr 27;292(5517):727-30.
112. Häcki J, Egger L, Monney L, Conus S, Rossé T, Fellay I, Borner C. Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2. Oncogene. 2000 May 4;19(19):2286-95.
113. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K, Tsujimoto Y, Tohyama M. Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol. 2004 May 10;165(3):347-56. Epub 2004 May 3.
114. Tadros SF, D'Souza M, Zhu X, Frisina RD. Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea. Apoptosis. 2008 Nov;13(11):1303-21.


------------------------------------------------------------------------ 第 16 筆 ---------------------------------------------------------------------
系統識別號 U0007-0507201010190100
論文名稱(中文) 不同形式神經生長相關蛋白 zGAP-43S41A、zGAP-43S41D 和 zGAP-43WT 對於斑馬魚胚胎神經發育和運動行為影響之研究
論文名稱(英文) The Effect of zGAP-43S41A, zGAP-43S41D or zGAP-43WT in Developing Neurons and Animal Behavior in Zebrafish Embryo
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 林育昌
學號 M120097033
學位類別 碩士
語文別 中文
口試日期 2010-06-25
論文頁數 49頁
口試委員 委員-李怡萱
委員-李明亭
指導教授-周志銘
關鍵字(中) GAP-43
關鍵字(英) GAP-43
學科別分類
中文摘要 神經生長相關蛋白(生長相關蛋白43;的GAP - 43 )是一種專一性在神經細胞表達的蛋白,當神經細胞進行發育或再生時,的GAP - 43蛋白會高度分佈於神經細胞的生長錐;的GAP -43蛋白,會促進神經細胞突觸的生長與神經細胞的可塑性,而且在神經傳導和突觸的生長也扮演重要的角色,而神經訊息的傳導會影響動物的運動行為。本研究,為了探討不同形式突觸生長相關蛋白的GAP - 43對於動物運動行為的影響,我們利用了雙於克啟動子,分別去調控目標基因( zGAP - 43S41A , zGAP - 43S41D或zGAP - 43WT )和報導基因(綠色螢光蛋白, GFP)的共同表達於發育時期的斑馬魚神經細胞,結果顯示,不同形式zGAP - 43轉殖的斑馬魚,皆會造成神經纖維生長排列不規則,而且在斑馬魚運動行為研究中發現, zGAP - 43S41A轉殖的斑馬魚相較於野生型對照組,在觸覺刺激結果顯示具有較短的移動距離而且在尾部刺激結果顯示會有較長的移動時間,而在zGAP - 43S41D轉殖斑馬魚同樣的刺激條件,則顯示相反的結果。由本研究結果推測zGAP - 43S41A轉殖斑馬魚,在觸覺刺激後移動反應能力降低的結果,可能是由於不活化的zGAP - 43S41A蛋白,影響神經纖維不正常排列與生長,進而影響神經訊息傳遞所造成。
英文摘要 生長相關蛋白43(的GAP - 43 )是一種神經元特異性蛋白,高效表達,在神經生長錐在開發和再生。這是導致軸突延長,突觸可塑性和突觸傳遞的神經系統。此外,動物的行為是依賴於神經迴路。為了排除這種可能性的軸突延伸和突觸傳遞有關的GAP - 43規定的模式動物的行為。在這項研究中,我們使用雙啟動子驅動的zGAP zHuC - 43S41A , zGAP - 43S41D或zGAP - 43WT基因與綠色熒光蛋白基因表達的記者在神經系統在斑馬魚發育階段。過表達zGAP - 43S41A , zGAP - 43S41D或zGAP43WT在斑馬魚胚胎形態的缺失造成了一些混亂和在神經軸突的延伸。通過結合觸摸檢測與觀察游泳能力,觸摸靈敏度zGAP - 43S41A , zGAP - 43S41D和zGAP - 43WT轉基因斑馬魚均高於野生型對照。游泳能力的觀察結果表明, zGAP - 43S41D轉基因斑馬魚有更多的游泳能力比zGAP - 43S41A轉基因斑馬魚和野生型對照。觸覺刺激後,在zGAP - 43S41A轉基因斑馬魚少了游泳的能力比野生型對照。在這項研究中,我們演示過表達zGAP - 43S41A , zGAP - 43S41D或zGAP - 43WT ,受影響的觸摸響應和游泳的能力蘊涵的狀態中GAP - 43的影響軸突延伸和突觸傳遞,以規範的模式動物行為。
論文目次 縮寫表...................我
圖表目錄.................二
中文摘要.................三
英文摘要.................四
壹,前言................. 1
貳,材料與方法........... 8
參,結果................. 15
肆,討論................. 21
伍,參考文獻............. 23
陸,結果圖表............. 28
參考文獻 艾爾哈特,美兆,李,衛生署,威爾遜,運輸署,米勒,BE大會,米勒,明尼蘇達和Skalko和RG ( 2007年)。運動失調和神經化學變化,沐浴後的斑馬魚幼蟲暴露於氟西汀(百憂解) 。 Neurotoxicol Teratol 29( 6): 652-664 。
Arrenberg , AB公司,德爾拜奈,樓及拜爾,譯( 2009) 。光控與halorhodopsin斑馬魚的行為。姜麗萍,科學的PRoC Natl美阿106( 42) : 17968-17973 。
巴斯, GS認證,雅各布森,研發,維拉格,一,席林, j的與謝訥, JH ( 1987)。一級結構和基因轉錄調控中GAP - 43 , 1蛋白與神經生長有關。細胞49( 6): 785-791 。
偶然發現,李,佩羅內,比佐澤羅,倪&芬克勒施泰因,警司( 1987)。分子特性的生長相關蛋白的GAP - 43(乙- 50) 。 J神經化學48( 5): 1640年至1647年。
Biffo ,南, Verhaagen , j的, Schrama , LH的, Schotman ,體育, Danho之際,與馬戈利斯,佛羅里達州( 1990年)。 B-50/GAP43表達與進程的產物在小鼠胚胎神經系統。歐元J神經科學雜誌2(6 ) : 487-499 。
Brustein ,大腸桿菌,馬蘭迪,北路,科瓦利丘克,士元,德拉波,體育及Konnerth ,答:( 2003)。 “在體內“神經元網絡活動的監測,斑馬魚的雙光子鈣(2 + )成像。 Pflugers拱446( 6): 766-773 。
卡羅尼,體育,愛格納湖與施耐德,三( 1997年) 。局部神經調節的內在因素和突觸突觸外生長在成年神經肌肉交界處。細胞與外來的動物136( 3): 679-692 。
查普曼,急診室,金,博士,亞歷山大,嘉,尼科爾森,電訊管理局局長及風暴博士( 1991年)。鈣調蛋白結合的特性域neuromodulin 。功能意義絲氨酸41和苯丙氨酸42。 J Biol Chem的266 ( 1): 207-213 。
克拉克,法學博士,海斯,英國石油公司,亨特,警司和羅伯茨,答:( 1984) 。感官生理學,解剖學,免疫組織化學的Rohon鬍子神經元的爪蟾胚胎。 J生理學348: 511-525 。
科金斯,第j的&茲維爾斯,譯( 1989)。證據單一蛋白激酶C介導的磷酸化位在大鼠腦組織蛋白的B - 50。 J神經化學53( 6): 1895年至一九零一年。
德糧食裝卸,生產通知書,麵包車Hooff ,一氧化碳,蒂莉,不列顛哥倫比亞省, Oestreicher ,AB型, Schotman ,體育及Gispen炎( 1985)。磷的B - 50在神經生長錐從胎鼠大腦。神經科學萊特61( 3): 235-241 。
丹尼, j的乙( 2006年)。分子機制,生物學行為和神經藥理學的生長相關蛋白的GAP - 43 。柯Neuropharmacol 4( 4): 293-304 。
多諾萬, SL和Mamounas ,洛杉磯,安德魯斯,上午,藍,是我和麥卡斯蘭, js中( 2002年)。的GAP - 43的正常發展是至關重要的前腦血清素神經支配。 J神經科學雜誌22( 9): 3543-3552 。
道格拉斯,廣告, Kraves ,南, Deisseroth ,光, Schier ,自動對焦和恩格特,樓(2008年) 。逃逸行為引起單, channelrhodopsin - 2在斑馬魚的體感誘發神經元的高峰。柯測定儀18( 15): 1133年至1137年。
弗雷,博士,勞克斯,噸,徐湖,施耐德,正與卡羅尼,第(2000 ) 。共享和獨特的作用CAP23在肌動蛋白基因GAP43和調控,神經軸突生長和解剖可塑性。細胞與外來的動物149( 7): 1443年至1454年。
甘比,三,瓦格,三菱商事,阿倫和RG & Baizer湖( 1996年)。生長相關蛋白43(的GAP - 43 )促進荷爾蒙分泌肽在小鼠垂體前葉瘤AtT - 20細胞。與 J Biol Chem 271( 17): 10023-10028 。
Gispen ,白盔部隊, Leunissen ,1997, Oestreicher ,AB型, Verkleij ,歐塞爾和茲維爾斯,譯( 1985)。突觸前定位的B - 50磷酸化:在(促腎上腺皮質激素)敏感的蛋白激酶底物參與了大鼠腦polyphosphoinositide代謝。腦水庫328( 2): 381-385 。
他,問,登特,大腸桿菌W.及美日,法光樓( 1997年)。調節肌動蛋白絲行為的間隙- 43( neuromodulin )是依賴於絲氨酸的磷酸化狀態41 ,蛋白激酶C的網站。 J神經科學雜誌17( 10): 3515-3524 。
Higashijima ,南,馬西諾,馬,曼德爾灣及Fetcho和JR ( 2003)。影像在斑馬魚神經元的活動行為,這是基因編碼的鈣指標。神經生理學90( 6): 3986-3997 。
奧克米耶,米,維特里,南,比古,南,布魯爾, j的,奧塞伊, j的與聽,子( 2010年) 。基因GAP43表達和增強軸突生長的粘多醣貯積症IIIB型皮層神經元的文化。 J神經科學水庫88( 1): 202-213 。
胡拉多,洛杉磯, Chockalingam ,聚苯乙烯&哈雷特,硬件( 1999年)。 Apocalmodulin 。生理學牧師79 ( 3): 661-682 。
克魯格,光,譚,因為,路,長及Sretavan ,德國之聲( 1998年)。視網膜神經節細胞軸突進展從視交叉視束開始發展需要自主功能的細胞中GAP - 43 。 J神經科學雜誌18( 15): 5692-5705 。
Kusik ,體重,哈蒙德博士和Udvadia ,歐塞爾(2010年) 。轉錄調控區域發展需要的基因GAP43和再生視網膜神經節細胞。開發注射Dyn 239( 2): 482-495 。
勞克斯,噸,深見,光,爾倫,米,戈盧布,噸,弗雷,四及卡羅尼,第(2000 ) 。基因GAP43 ,馬克斯,有價證券和CAP23調製(4,5 )芘(2 )在質膜排,調節皮層肌動蛋白細胞通過一個共同的動力機制。細胞與外來的動物149( 7): 1455年至1472年。
梁,十,魯,士元,內伯特,電訊管理局局長及雷什,醫學博士( 2002年)。質譜分析GAP-43/neuromodulin揭示了存在的各種脂肪酸酰化種。與 J Biol Chem 277( 36): 33032-33040 。
低,硒,週之際, ,忠,一,聖情人湖,斯普拉格,釤,平田,閣下,崔,第一次世界大戰,休謨,埃古桑田,九年(2010年) 。娜(五)需要1.6A的是正常啟動電機電路通常由觸覺刺激興奮。開發神經生物70( 7): 508-522 。
邁爾,DL,各摩尼,南,多諾萬, SL和Soppet ,博士, Tessarollo湖,麥卡斯蘭, js中與美日, KF法( 1999年)。中斷的情況下皮質和皮質地圖桶生長相關蛋白( GAP)的-43基因敲除小鼠。姜麗萍,科學的PRoC Natl美阿96( 16): 9397-9402 。
美日,氟化鉀, Hammang ,太平紳士,登特,電子戰與貝特格,電子工程( 1996年)。誘變ser41為 Ala抑制協會的GAP - 43與膜骨架中GAP - 43 -細胞缺陷PC12B :對細胞粘附和細胞骨架的組成和膜的突起。 J神經生物29( 2): 213-232 。
阮湖,他,問及美日,法光樓(2009年) 。規例中GAP - 43在41絲氨酸充當一個開關來調節其內在和外在的行為日益神經元,通過改變膜的分佈。分子細胞神經科學41( 1): 62-73 。
公園,高等法院,金,甲烷,裴,蔡耀君,姚照東,石蔭,金,上海,香港,水庫,善, j的,柳,千瓦, 々 ,米,平野,噸,三木,北路, Chitnis , AB公司與許,熱釋光(2000 ) 。分析上游要素的於克子導致建立轉基因熒光斑馬魚神經元。開發外來的動物227( 2): 279-293 。
選手佩特里,噸,馬納洛,大腸桿菌,瑞安, j的,聖阿芒湖及沃什伯恩,第(2009 ) 。谷氨酸驅動器的觸摸響應通過在頭端循環脊髓斑馬魚的胚胎。開發神經生物69( 12): 780-795 。
雷耶斯,河, Haendel ,米,格蘭特,博士,梅蘭科,大腸桿菌及艾森, js中( 2004年)。斑馬魚Rohon慢變性,比爾德在程序性細胞死亡的神經元。開發注射Dyn 229( 1): 30-41 。
魯滕貝格,甲,主題 2 ,扎富托,南,塞拉諾,體育及南宮,美國( 2000年)。後基因表達增強學習的大腦發育的蛋白質。姜麗萍,科學的PRoC Natl美阿97( 13): 7657-7662 。
聖情人湖及德拉波,第(2000 ) 。運動神經元活動模式有關的行為的斑馬魚早期胚胎。 J神經科學雜誌20( 11): 3964-3972 。
桑托內托,閣下,馬丁斯,歐塞爾, Minatel ,大腸桿菌及馬凱斯,兆焦耳( 2003年)。在MDX小鼠軸突發芽及其與細胞療法和基因介導的杜氏肌營養不良症。神經科學萊特343( 1): 67-69 。
施賴爾,四j的與謝訥,瑞星( 1991)。命運的GAP - 43升的81.67脊髓軸突周圍神經損傷:延遲的積累和相關性再生的潛力。 J神經科學雜誌11( 12): 3738-3751 。
沈士元,摩尼,南,多諾萬, SL和施沃布,日本腦炎及美日, KF法( 2002年)。生長相關蛋白- 43是需要連合軸突指導發展脊椎動物的神經系統。 J神經科學雜誌22( 1): 239-247 。
許,財政司司長, Azmitia ,歐共體,馬沙克醫生,帕克,度Pj &魯滕貝格,答:( 1994)。膠質細胞源性重組S100B蛋白選擇性抑制β蛋白激酶C( PKC)的磷酸化的神經元特異性蛋白F1/GAP43 。腦水庫分子腦水庫21( 1-2) : 62-66 。
謝訥,瑞星和維拉格,一( 1989年)。翻譯後膜附著和動態脂肪酸酰化的神經生長錐的蛋白質,的GAP - 43 。細胞與外來的動物108( 2): 613-624 。
謝訥,瑞星&威拉德,米( 1981年)。 Axonally運輸與軸突生長相關蛋白在兔中樞和周圍神經系統。細胞與外來的動物89( 1): 96-103 。
斯特里特馬特,釤,克黑澤角,黃,特等,直下,閣下及菲什曼,三菱商事( 1995年)。尋路是不正常的神經元在小鼠神經生長錐缺乏蛋白質的GAP - 43 。細胞80( 3): 445-452 。
斯沃博達,雷克南,利納雷斯,自動曝光和貝拉, AB公司( 2001年)。活性調節細胞程序性死亡的斑馬魚Rohon鬍子神經元。發展128( 18): 3511-3520 。
索博陶,南,戈羅斯蒂薩,體育,德爾拜奈,樓Wyart角,福爾廷,DL,各科爾斯特德,科威特第納爾, Tulyathan ,澳, Volgraf ,米,沼,河,亞倫, HL的斯科特,克朗,克萊默,相對濕度,弗蘭納裡, j的,拜爾,閣下,特拉納,四及Isacoff ,安永( 2007年)。遠程控制的神經元活動與一輛輕型門控谷氨酸受體。神經元54( 4): 535-545 。
Udvadia ,甲譯( 2008) 。 3.6 kb的基因組序列暗紋東方能夠促進軸突生長相關基因表達的斑馬魚神經元的再生和發展。基因expr的模式8(6 ) : 382-388 。
Udvadia ,歐塞爾,科斯特, RW光碟及謝訥, JH (2001)。的GAP - 43啟動子在轉基因斑馬魚揭示了不同的信號,為中樞神經系統發育過程中軸突的生長和再生。發展128( 7): 1175年至1182年。
Vanselow , j的, Grabczyk ,大腸桿菌,平, j的, Baetscher ,米,鄧,南與菲什曼,三菱商事( 1994年)。的GAP - 43轉基因小鼠:分散的基因組序列賦予的GAP - 43樣在開發過程中的表達模式和再生。 J神經科學14( 2): 499-510 。
王j的,甘比爾,甲,麥克勞克林,第&默里,四( 2004年)。一個計算模型的靜電隔離的PI ( 4,5) P2的膜,吸附基本肽。 Biophys J 86( 4): 1969年至1986年。
威德默,樓及卡羅尼,第(1993 ) 。磷酸現場誘變的生長相關蛋白的GAP - 43對細胞調控的影響和傳播形態。細胞與外來的動物120( 2): 503-512 。
威廉姆斯,司法機構政務長,巴里奧斯,甲, Gatchalian角,魯賓,黃瓜,威爾遜,西南及持有人,注( 2000年)。程序性細胞死亡中的斑馬魚rohon鬍子神經元是受TrkC1/NT-3信號。開發外來的動物226( 2): 220-230 。
吳,閣下,熊之際,正與美湖(2010年) 。要建設一個突觸:信號轉導通路在神經肌肉接頭組裝。發展137( 7): 1017至1033年。
Wyart角,德爾拜奈,樓,翹曲,大腸桿菌,斯科特,克朗,特拉納,博士,拜爾,閣下及Isacoff ,安永(2009年) 。 Optogenetic解剖一個行為模塊的脊椎動物脊髓。自然461( 7262 ) : 407-410 。
朱熹,士元,瑞安, j的,高貴,南,餘米, Yilbas ,自動曝光和克爾,米(2010年) 。受損的多巴胺能神經元發育和運動功能的斑馬魚的損失pink1功能。歐元J神經科學雜誌31( 4): 623-633 。
張士元,博城,十, Schoepfer河, Holtmaat ,歐塞爾, Verhaagen , j的,埃姆森,電腦,利伯曼,氬和安德森,生產通知書( 2005年)。生長相關蛋白GAP - 43和母語協同行動,以促進再生軸突生長的浦肯野細胞在體內。姜麗萍,科學的PRoC Natl美國102 (41 ) : 14883-14888 。
祖伯,的MX ,斯特里特馬特,釤及菲什曼,三菱商事( 1989年)。甲膜定位信號的氨基端的神經元蛋白的GAP - 43 。自然341( 6240 ) : 345-348 。

------------------------------------------------------------------------ 第 17 筆 ---------------------------------------------------------------------
系統識別號 U0007-0507201014501400
論文名稱(中文) 穿心蓮內酯誘發蛋白磷酸酯酶2A抑制內毒素/干擾素- γ誘發轉錄因子NFκB的活化之分子機轉探討
論文名稱(英文) 抑制機制的誘導蛋白磷酸酶2A條穿心蓮內酯對 LPS / IFN -γ的誘導NFκB的激活
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 陳璿文
學號 M120097017
學位類別 碩士
語文別 中文
口試日期 2010-07-01
論文頁數 72頁
口試委員 委員-黃德富
委員-顏茂雄
委員-楊春茂
共同指導教授-許準榕
指導教授-馮琮涵
關鍵字(中) 穿心蓮
蛋白去磷酸酶
關鍵字(英) andrographolide
protein phosphatase 2A
NFκB
學科別分類
中文摘要 近年來的研究指出,一種穿心蓮屬的植物萃取物成分穿心蓮內酯(穿心蓮)可以藉由抑制轉錄因子NFκB的達到抗發炎的效果,而穿心蓮如何調控NFκB的訊息傳遞路徑與機制仍須深入探討。本篇論文發現穿心蓮內酯在大鼠血管平滑肌細胞( VSMC的)對於脂多醣體(LPS)刺激與干擾素γ (干擾素- γ)所刺激的誘導型一氧化氮合成酶( iNOS)的生成具有抑制的的效果,進一步探討穿心蓮對於iNOS的上游NFκB的訊息傳遞路徑的調控,例如P65蛋白的磷酸化與轉位結合的DNA活化的NFκB的能力是否與蛋白去磷酸酶酶2A(PP2A )有關。為了探討PP2A的對於NFκB的活化所扮演的角色,本篇PP2A的抑製劑論文使用岡田酸(OA)以及中性鞘磷脂酶(中性鞘磷脂酶, nSMase )抑製劑3 - O -甲基鞘磷脂( 3中耳炎)後,發現辦公自動化和3 -中耳炎會抑制穿心蓮對於內毒素/干擾素- γ刺激造成的iNOS的表現。此外本篇論文也發現在大鼠血管平滑肌細胞中,穿心蓮會增加PP2A的活性以及細胞中神經醯胺(神經酰胺)的含量。從實驗結果推論,穿心蓮可能藉由活化nSMase -神經酰胺- PP2A的訊息路徑,使P65ser536去磷酸化,導致的NFκB的不活化,進而影響下游iNOS表達的生成。
英文摘要 最近的研究表明,轉錄因子核因子(NF )- κB的抑制可能有助於保護抗炎作用的穿心蓮內酯,豐富的組成部分屬植物穿心蓮。但是,確切的機制,穿心蓮內酯抑制NF -κB的信號仍不清楚。因此,我們在參與調查的機制,穿心蓮內酯抑制NF -κB的信號在大鼠血管平滑肌細胞(VSMC )受炎症刺激,脂多醣( LPS)和干擾素(IFN )- γ 。穿心蓮內酯是能抑制內毒素/干擾素-γ誘導的誘導型一氧化氮合酶( iNOS)的表達對大鼠血管平滑肌細胞。穿心蓮內酯也可以抑制LPS / γ-干擾素誘導p65的核移位, DNA結合活性, p65的Ser536磷酸化和NF -κB的記者活動。穿心蓮內酯抑制這些行動可以預防由岡田酸,蛋白磷酸酶2A ( PP2A的)抑製劑,或PP2A的小干擾 RNA 。此外,3 - O -甲基鞘磷脂(三甲酯) ,中性鞘磷脂酶( nSMase )抑製劑,也能抑制穿心蓮行動在LPS / IFN -γ的刺激血管平滑肌細胞。此外,穿心蓮被證明PP2A的活性增加血管平滑肌細胞。神經酰胺在細胞水平也升高,血管平滑肌細胞暴露於穿心蓮。兩者合計,這些結果表明,穿心蓮內酯可能導致nSMase -神經酰胺, PP2A的級聯活化p65的Ser536去磷酸,導致NF -κB的失活和隨後的大鼠血管平滑肌細胞中iNOS的表達下調LPS刺激和IFN - γ 。
論文目次 目錄一
中文摘要2
英文摘要3
縮寫對照表4
壹,緒論5
貳,材料與方法20
參,結果32
肆,討論39
伍,結論44
陸,參考文獻45
柒,圖表56
捌,附錄70
參考文獻 阿德利,米和鮑德溫,甲南2006。 IKK-i/IKKepsilon控制構,腫瘤細胞相關因子- κB的活性通過調節絲氨酸- 536 p65/RelA磷酸化。 J Biol Chem的281: 26976-84 。

Bakkar ,北部&加特里奇,直流2010年。核因子- κB的信號:一個故事的兩種途徑在骨骼肌肉生成。生理學牧師90: 495-511 。

寶, z的,關,南,鄭正,吳,南,黃,上海,凱梅尼,糖尿病,梁,英國石油公司和黃,是2009年。一種新型的抗炎作用穿心蓮內酯通過抑制哮喘的核因子-κB途徑。上午J Respir危重病急救醫學179: 657-65 。

巴恩斯,第j的和卡琳, 1997米。核因子- κB的:一個關鍵的轉錄因子在慢性炎症疾病。 ñ英格蘭醫學雜誌336 : 1066年至1071年。

鮑爾, j的,於伊角, Brenmoehl , j的,奧伯邁爾,樓及博克, j的2009年。基質金屬蛋白酶誘導白細胞介素1表達-1β需要酸性鞘磷脂酶。 FEBS萊特583 : 915-20 。

貝爾蒂尼,一,卡爾德龍,五, Fragai ,米, Luchinat角及塔盧,大腸桿菌2009。結構基礎上,絲氨酸/蘇氨酸磷酸酶抑制典型的小分子斑蝥素及去甲斑蝥。醫志化學52: 4838-43 。

Bonizzi ,葛&卡琳, 2004米。這兩個核因子- κB的激活途徑,其作用在先天免疫和適應性免疫。趨勢免疫25: 280-8 。

品牌,光,頁,南, Rogler ,克,巴茨奇,甲,布蘭德,河, Knuechel河,佩奇,米, Kaltschmidt角,博伊爾勒,巴勒斯坦權力機構和諾伊邁爾,四1996。激活轉錄因子核因子-κB在動脈粥樣硬化存在病變。 J萬方數據投資97: 1715年至1722年。

科比,賽馬,韋斯特法爾, RS和Wadzinski , 1999年。甲基化的C -末端的亮氨酸殘PP2A的催化亞基是很重要的具有約束力的監管Balpha亞基。生化J 339(鉑2): 241-6 。

巴斯,閣下,多莉,甲,施米茨,毫升,弗蘭克,河,利文斯通,米,雷希,光及克拉赫特, 2004米。磷酸化絲氨酸468由葛蘭素史克公司, 3beta基礎p65的負調控因子- κB的活性。 J Biol Chem的279 : 49571-4 。

黃油,勞工處,斯普林爾博士,切斯特,啊,埃文斯的TJ ,斯坦德,恩, Parums ,攝像機,雅各布,氫,波拉克, JM 1996。誘導型一氧化氮合成酶是存在於人類,促進動脈粥樣硬化病變的形成和活動的過氧化亞硝酸鹽。實驗室投資75: 77-85 。

卡拉布雷斯角,伯爾曼,上海, Babish ,漏電流,馬,十,神道,黃瓜,多爾,米,水井,光,溫納,加利福尼亞&斯坦迪什,洛伊2000。 I期試驗穿心蓮內酯在HIV陽性患者和正常人。 Phytother水庫14: 333-8 。

Chandrasekar ,乙, Mummidi ,南, Mahimainathan湖,帕特爾,糖尿病腎病,貝利,鍶,伊瑪目,深圳,格林,廁所和瓦倫特,歐塞爾2006。白細胞介素18誘導人冠狀動脈平滑肌細胞遷移依賴於核因子-κB和AP - 1的介導基質金屬蛋白酶9的表達,並抑制了阿托伐他汀。 J Biol Chem的281: 15099-109 。

陳前,張陛下,許兆焦耳,胡適,醫藥,趙一匯,趙,野生型,林, 2009年甲烷。肽聚醣誘導COX - 2表達在巨噬細胞的激活中性鞘磷脂酶,神經酰胺通路。 J Biol Chem的284: 20562-73 。

陳, j的,馬丁,乙屬及布勞提根,四屬1992年。調節蛋白質絲氨酸蘇氨酸磷酸酶型酪氨酸磷酸甲。科學257: 1261-4 。

週,賽馬,青年,德國之聲, Golenbock ,藥物療法,基督, WJ通訊及Gusovsky , 1999號。 Toll樣受體4介導脂多醣誘導的信號轉導。 J Biol Chem的274 : 10689-92 。

克勞夫斯,答W. ,裡迪,肌肉萎縮症及克勞夫斯,米1983米。機制動脈損傷後狹窄。實驗室投資49: 208-15 。

庫恩,嘉理及恩斯特,大腸桿菌2004。穿心蓮在治療上呼吸道感染:一個系統的安全性和有效性審查。普蘭塔醫學70: 293-8 。

多芬,脊髓肌肉及考爾尚,答: 2006年。內皮細胞內毒素信號。實驗室投資86: 9-22 。

Detmers ,功率放大器,埃爾南德斯,米,馬奇特, j的,海辛,閣下,伯頓角,蒙特,南,春,南,弗萊徹,博士,卡,播音員, Lisnock , j的,韋克爾,河,貝格斯特羅姆,法學博士, Shevell ,教育署署長,赫爾馬諾夫斯基-沃薩特卡,甲,麻雀,粗蛋白,趙,伊蘇,雷達,道瓊斯,賴特,政府統計處及純,大腸桿菌2000。不足之處誘導型一氧化氮合酶在動脈粥樣硬化導致降低載脂蛋白E基因缺陷小鼠。 J免疫165: 3430-5 。

多爾蒂,商標, Asotra ,光,菲茨帕特里克,洛杉磯,喬石, JH ,威爾,播音員, Detrano ,鋼筋混凝土,鄧斯坦,鉻,沙和PK & Rajavashisth ,結核病2003。鈣化的動脈粥樣硬化:骨生物學和慢性炎症在動脈十字路口。姜麗萍,科學的PRoC Natl美阿100: 11201-6 。

杜蘭,甲,迪亞茲-梅科,林茂及莫斯卡特, j的2003年。基本釐清的Ser311磷酸化作用的zetaPKC在NF -κB的轉錄激活。歐洲分子生物學組織J 22: 3910-8 。

Erridge角,貝內特,格雷羅,大腸桿菌及Poxton ,紅外2002。內毒素的結構和功能。微生物感染4: 837-51 。

芬頓,米j的& Golenbock , 1998年四噸。脂多醣結合蛋白和受體。 J Leukoc測定儀64: 25-32 。

Forstermann ,美國,克洛斯,指引波洛克, js中,中根,米,施瓦茨,體育,迦特,一&克萊納特,閣下1994年。一氧化氮合成酶同工酶。表徵,純化,分子克隆和功能。高血壓23: 1121年至1131年。

弗朗茲,南,埃特爾,葛&鮑爾扎克斯, j的2007年。機制的疾病: Toll樣受體在心血管疾病。納特萬方數據Pract中西醫結合學報4: 444-54 。

弗朗茲,南,胡法光,拜耳,乙, Gerondakis ,南, Strotmann , j的,阿達梅克,甲,埃特爾,葛&鮑爾扎克斯, j的2006年。缺席的NF -κB的亞單位的P50改善心肌梗死後心力衰竭。 FASEB J 20: 18年至1920年。

富思特,五, 1994年。劉易斯答康納紀念講座。導致心肌梗死的機制:從研究的見解血管生物學。流通90: 2126-46 。

加利斯, z的南與Khatri ,執中2002。基質金屬蛋白酶在血管重塑及動脈粥樣硬化:好的,壞的,醜陋的。保監會水庫90: 251-62 。

戈什,南與卡琳, 2002米。失踪件在NF -κB的難題。電池109增刊: S81 - 96 。

灰色,體育W. & Goeddel ,四五, 1982年。結構人體免疫干擾素基因。自然298: 859-63 。

Hannun ,士元答: 1994年。該鞘磷脂週期和第二信使的功能神經酰胺。 J Biol Chem的269: 3125-8 。

漢森,葛光2005。炎症,動脈粥樣硬化和冠心病。 ñ英格蘭醫學雜誌352: 1685年至1695年。

哈維, j的大腸桿菌,李,北部&拉姆吉, 2007年四頁。關鍵作用酪蛋白激酶2和磷酸肌醇-3激酶在干擾素-γ誘導單核細胞趨化蛋白的表達- 1和其他關鍵基因在動脈粥樣硬化有牽連。動脈硬化雜誌凝血酶Vasc測定儀27: 806-12 。

哈維, j的大腸桿菌及拉姆吉,四頁2005。干擾素γ和動脈粥樣硬化:親或反動脈粥樣硬化? Cardiovasc水庫67: 11-20 。

伊達爾戈,馬,羅梅羅,甲,菲格羅亞, j的,科爾特斯,體育,鼻甲,二, Hancke , JL &布爾戈斯,類風濕性關節炎2005。穿心蓮內酯干擾具有約束力的核因子-κB的DNA在HL - 60衍生的中性粒細胞。溴J藥理學144: 680-6 。

許兆焦耳,張對照,陳,三菱商事,陳,公元前,馬,惠普,宏, CY和林,甲烷凋亡信號調節激酶1肽誘導的COX - 2表達在巨噬細胞。 J Leukoc測定儀87: 1069年至1082年。

許兆焦耳,許,小,林,甲烷,沉,我的&許,賽揚線粒體機制澱粉樣β肽引起的腦血管病變。 Biochim Biophys文獻1800: 290-6 。

詹森斯,五及戈里斯, j的2001年。蛋白磷酸酶2A :高度管制的家族絲氨酸/蘇氨酸磷酸酶在細胞生長和牽連的信號。生化J 353: 417-39 。

吉米,大腸桿菌,青木,光,齊藤,閣下,德阿奎斯托,樓,5月,美兆,中村,一,須藤,噸,小島,噸,岡本,樓,福島,閣下,岡部,光,大谷,光及高希,第2004。選擇性抑制NF -κB的塊破骨細胞生成和防止骨質破壞體內炎症。納特醫學10: 617-24 。

卡琳,米及格雷滕,埃弗阿爾2005。核因子- κB的:連接炎症和癌症的免疫能力的發展和進步。納馮免疫5: 749-59 。

卡琳,米,林, A.,2002年。核因子-κB在生與死的十字路口。納特免疫3: 221-7 。

勝田,南與加地亮, 2003噸。動脈粥樣硬化和細胞外基質。 J動脈粥樣硬化凝血酶10: 267-74 。

金穗,南, Kurosinski ,體育,尼特奇,馬幣及郭茨, j的2003年。激活的ERK和JNK信號通路所造成的神經元特異性抑制PP2A的轉基因小鼠。上午病理學期刊163: 833-43 。

克萊納特,閣下,帕茨,甲,連接器,光與施瓦茨,下午2004。規例中的表達誘導型一氧化氮合酶的影響。歐元J藥理學500: 255-66 。

Kolesnick河,北部& Kronke , 1998米。調節神經酰胺生產和凋亡。安奴馮生理學60: 643-65 。

Lehrke ,米,悲哀,米, Broedl ,加州大學,萊布赫斯角,勞本德爾,反相,貝克爾,甲,馮齊格勒樓Tittus , j的,賴澤,米,貝克爾,長,鄉家灣,斯坦貝克,克,萊伯,仙及Parhofer , 2009公斤。 MMP - 1的血清水平預測冠狀動脈粥樣硬化的人類。 Cardiovasc Diabetol 8: 50。

勒利奧,北路,凱維永-謝呂埃爾,南,索列爾,一,德拉謝拉- Gallay ,白細胞介素, Collinet ,乙,格拉耶,米,布多,光, Bettache ,北路, Poupon ,甲,雅南, j的和麵包車Tilbeurgh ,閣下2004年。甲基轉移酶的結構蛋白磷酸酶1( PPM1 ) ,一亮氨酸的羧基甲基化參與調控蛋白磷酸酶2A活性。 J Biol Chem的279 : 8351-8 。
李南,王湖,伯爾曼,馬,張士元和多爾夫, ME的2006年。 RNA干擾對小鼠星形膠質細胞識別屏幕磷酸酶的調節因子- κB的信號。分子細胞24: 497-509 。

盧西斯,答j的2000年。動脈粥樣硬化。自然407 : 233-41 。

馬赫,樓Schonbeck ,美國,邦內福伊,九年, Pober , js中與利比,第1997。活化的單核/巨噬細胞功能相關的急性動脈粥樣硬化並發症結紮CD40分子:感應膠原酶,基質分解素,及組織的因素。流通96: 396-9 。

馬歇爾,米, Anilkumar ,北路,萊蘭, j的,沃克,律政司司長,肯蒂什,賽馬,沙阿,上午及洞穴,交流2009年。蛋白磷酸酶2A有助於心功能不全引起的內毒素血症。 Cardiovasc水庫82: 67-76 。

Mattioli ,一,錫巴爾德,甲,布赫爾角,查爾斯,反相,中野,閣下,土井,噸,克拉赫特,米和施密茨, 2004毫升。瞬態和選擇性核因子-κB p65的磷酸化絲氨酸536誘導T細胞介導的共刺激是我κB的蛋白激酶的動力學測試和控制的p65的核進口。 J免疫172: 6336-44 。

麥凱勒,通用電氣,麥卡里,德國之聲,薩塔爾,北部&麥克因尼斯,興業2009年。腫瘤壞死因子在動脈粥樣硬化的作用?自身免疫性疾病的經驗教訓。納馮Cardiol 6: 410-7 。

馬比,米長和沃爾特克1993。蛋白絲氨酸/蘇氨酸磷酸酶:結構,規制,在細胞生長和功能。生理學牧師73: 673-99 。

南杜裡,南, Nyavanandi ,唯, Thunuguntla ,不袗,卡蘇,南, Pallerla ,旺角,羊,聚苯乙烯,拉賈格帕蘭,南,庫馬爾,類風濕性關節炎,拉曼努金河,巴布, JM , Vyas ,光,黛維,因為,雷迪,開始與於阿克拉, 2004年五。合成和構效關係中的穿心蓮內酯類似物作為新的抗癌藥物。 Bioorg醫學化學萊特14: 4711-7 。

彌敦道,謝正和,問: W. 1994。一氧化氮合成酶:作用,通行費,和控制。單元78: 915-8 。


尼什,因為,葛維寶,在曾,閣下,楊,安,霍伯特,我, Karmali ,五,饒,為&馬達拉, JL 2000。原核反應的調節上皮抑制IkappaB的-α泛素化。科學289: 1560-3 。

歐文斯,葛光1995。分化調節血管平滑肌細胞。生理學牧師75: 487-517 。

帕洛薩里,閣下,邊寧頓,終審法院首席法官, Larmas ,米,愛德華茲醫生, Tjaderhane湖和薩洛, 2003噸。基因表達譜基質金屬蛋白酶( MMPs)和組織基質金屬蛋白酶抑製劑在人成牙本質細胞和成熟的牙髓組織。歐元J口腔科技111: 117-27 。

拉多姆斯基,米W. 1995。一氧化氮:生物介質,調製器和效應。安地中海27: 321-9 。

拉賈格帕蘭,南,庫馬爾,類風濕性關節炎, Deevi ,副秘書長,薩蒂亞納拉亞納角及拉賈戈帕蘭河2003。穿心蓮內酯,一種潛在的腫瘤治療劑分離穿心蓮。 J進出口有腫瘤學3: 147-58 。

Rajavashisth ,結核病, Andalibi ,甲,泰里托,三菱商事,柏林,司法機構政務長,納瓦卜,米,福蓋爾曼,上午及盧西斯,歐塞爾1990。誘導內皮細胞和巨噬細胞表達粒細胞集落刺激因子的修飾低密度脂蛋白。自然344: 254-7 。

雷伯湖,韋爾默朗,玉米,黑格曼,葛&弗羅薩,北路2009。 Ser276磷酸化的NF - kB的p65的由MSK1控制SCF的表達在炎症。公共科學圖書館之一4: e4393 。

羅斯河1999。動脈粥樣硬化-一種炎症性疾病。 ñ英格蘭醫學雜誌340: 115-26 。
魯伊斯-阿爾蓋洛, MB的,巴薩內斯,灣,孔塞洛,調頻和阿隆索,答: 1996年。不同的影響酶生成的神經酰胺和diacylglycerols在磷脂膜融合和洩漏。 J Biol Chem的271: 26616-21 。

魯沃格,第2001頁。神經酰胺通過調節細胞內的平衡不同的壓力信號通路。白血病15: 1153年至1160年。

魯沃格,第2003頁。細胞內的信號轉導通路激活的神經酰胺及其代謝物。藥理學水庫47: 383-92 。

Rzucidlo ,電磁,馬丁,嘉&鮑威爾接口,RJ 2007。調節血管平滑肌細胞的分化。 J Vasc外科45增刊答:答25 - 32 。

桑托羅,米灣,羅西,甲和阿米奇,長2003。核因子- κB和病毒感染:誰控制誰。歐洲分子生物學組織J 22: 2552-60 。

Schonbeck ,美國,馬赫,樓Sukhova ,競,墨菲,三,邦內福伊,九年, Fabunmi ,反相與利比,第1997。調節基質金屬蛋白酶的表達與血管平滑肌細胞的T淋巴細胞: 1作用CD40中的斑塊破裂?保監會水庫81: 448-54 。

施瓦茨,釤,坎貝爾,遺傳資源和坎貝爾, JH 1986。複製的血管平滑肌細胞的疾病。保監會水庫58: 427-44 。

森河和巴爾的摩, 1986年四。卡帕誘發的免疫球蛋白增強子結合蛋白核因子-κB通過翻譯後機制。單元47: 921-8 。

沉王耀,陳,正樓及邱,王唯2000。抑制大鼠中性粒細胞活性氧物種的產生和二萜內酯的附著力穿心蓮。普蘭塔醫學66: 314-7 。

沉王耀,陳,正樓及邱,王唯2002。防止氧自由基產生的穿心蓮內酯對人中性粒細胞:可能的機制(第)參與其抗炎作用。溴J藥理學135: 399-406 。

Shifera ,因為,弗里德曼, JM &霍維茨,碩士2008。 IKK的伽瑪( NEMO)的參與了協調的AP - 1和NF -κB的途徑。分子細胞生物化學310: 181-90 。

西尼奧雷利,體育,盧貝托角及Hannun ,士元答: 2001年。神經酰胺抑制NF -κB的激活涉及反向易位古典蛋白激酶C(PKC )同工酶:要求對激酶活性,羧基端磷酸化的蛋白激酶C的神經酰胺的反應。 FASEB J 15: 2401-14 。

薩默維爾,反相, Oblander ,南非和Apte和SS 2003。基質金屬蛋白酶:老狗新把戲。基因組測定儀4: 216。

明鏡周刊,第&美林,答:閣下, 1996年小。脂質代謝及細胞的生長規律。 FASEB J 10: 1388年至1397年。

斯塔裡,慧聰,錢德勒,AB型,迪恩斯莫爾,稀土,富思特,五, Glagov ,南,英薩爾之際,小,羅森菲爾德,我,施瓦茨,首席法官,瓦格納的WD &威斯勒, RW光碟1995。一個先進的類型定義動脈粥樣硬化和動脈粥樣硬化組織學分類。該委員會的一份報告對血管病變動脈粥樣硬化的會,美國心臟協會。動脈硬化雜誌凝血酶Vasc測定儀15: 1512年至1531年。

太陽之際,王,閣下,趙,十,餘士元,電風扇,士元,王十,魯,十張,葛富,南與楊, j的蛋白質磷酸酶2A作為一個有絲分裂原激活蛋白激酶激酶激酶3( MEKK3 )磷酸酶抑制溶血磷脂酸誘導IkappaB的激酶β/核因子- κB的激活。與 J Biol Chem 。

湯普森,法國興業,基納斯特, j的,派克,支持SD,哈維卡特,凡代樓及羅氏公司,JC 1995。止血因素和風險的心肌梗死或猝死患者心絞痛。歐盟協調行動的血栓形成和殘疾人心絞痛研究組。 ñ英格蘭醫學雜誌332 : 635-41 。

Thyberg , j的2002年。小窩和膽固醇分佈在血管平滑肌細胞表型的不同。 J組織化學Cytochem 50: 185-95 。

蒂瑟拉奇,肌肉萎縮症1999。一氧化氮在感染性休克。 Biochim Biophys文獻1411 : 437-55 。

托爾斯特赫,噸,李, j的, Vafai ,南與股票,巴頓2000。羧甲基化調節,通過控制磷蛋白磷酸酶2A協會監管乙亞基。歐洲分子生物學組織J 19: 5682-91 。

韋爾默朗湖,德王爾德,克,範達美,體育,萬登Berghe之際,與黑格曼, 2003克。轉錄激活的NF -κB的p65亞單位的有絲分裂原和應激活化蛋白激酶1( MSK1 ) 。歐洲分子生物學組織J 22: 1313年至1324年。

王,博士, Westerheide ,支持SD,漢森, JL &鮑德溫,因為, 2000年小。腫瘤壞死因子α誘導的Ser529磷酸化RelA/p65控制由酪蛋白激酶Ⅱ 。 J Biol Chem的275: 32592-7 。

汪湖,鄭, j的,都,士元,黃翼,李j的,劉灣,劉,終審法院首席法官,朱,士元,高,士元,徐,問,香港之際,與王,十軟骨低聚基質蛋白保持收縮表型血管平滑肌細胞相互作用與α( 7)測試( 1)整合。保監會水庫106: 514-25 。

王,體育,劉灣,歐,閣下,塘湖,羊, j的和唐, C. 1999年。一氧化氮合酶/一氧化氮途徑介導腸套疊發病的影響。中華醫學雜誌(英文版)112 : 1016-9 。

王系列YJ ,王,日本煙草,電風扇, QX型和耿民89 2007。穿心蓮內酯抑制核因子- kappaBeta激活和抑制內膜增生動脈再狹窄。細胞水庫17: 933-41 。

夏張遠芳,葉,燒烤,黎,耶穌青年會,王民89 ,何,許繼,林,十,瑤,十,馬,博士, Slungaard ,甲,赫貝爾,反相,鑰匙,生理鹽水及耕約格森米2004。穿心蓮內酯抑制炎症抑制NF - κB的活化,通過共價修飾減少半胱氨酸62的P50 。 J免疫173: 4207-17 。

楊, j的,電風扇,生長激素, Wadzinski ,BE大會,櫻井,閣下和里士滿,答: 2001年。蛋白磷酸酶2A與互動,直接去磷酸釐清的。 J Biol Chem的276: 47828-33 。

楊, z的,加加林,博士,聖洛朗,克, 3日,哈默爾,北路,托馬,一,胡錦濤,加州,岩佐,甲及麥卡弗里,助教2009。心血管炎症及病變細胞凋亡:一種新的連接通過干擾素誘導immunoproteasome 。動脈硬化雜誌凝血酶Vasc測定儀29: 1213-9 。

賢,崔京,許,賽揚,胡藝,陳,閣下,陳,西南,徐,李和j的, JM 2006。蛋白磷酸酶2A調節Bim表達通過Akt/FKHRL1信號通路在β-澱粉樣肽誘導的腦血管內皮細胞的死亡。 J神經科學雜誌26: 2290-9 。

趙灣,太陽湖,哈斯,米, Denenberg ,股份公司,黃,人力資源與尚利, 2008年總磷。 PP2A的調節上游成員的c - Jun氨基末端激酶絲裂原活化蛋白激酶信號通路。衝擊29: 181-8 。

鐘,閣下,福爾河大腸桿菌和戈什,第1998。磷酸化的NF - κB的p65的轉錄活性的PKA的刺激,促進新型雙價相互作用的共激活因子CBP/p300 。分子細胞1: 661-71 。

------------------------------------------------------------------------ 第 18 筆 ---------------------------------------------------------------------
系統識別號 U0007-0508201020372500
論文名稱(中文) 臺灣醫學專業素養展現情形之初探
論文名稱(英文) A Pilot Study of the Manifestation of Medical Professionalism In Taiwan
校院名稱 臺北醫學大學
系所名稱(中) 醫務管理學研究所
系所名稱(英) School of Health Care Administration
學年度 98
學期 2
出版年 99
研究生(中文) 陳盈盈
學號 M111097011
學位類別 碩士
語文別 中文
口試日期 2010-06-28
論文頁數 67頁
口試委員 委員-陳端容
委員-蔡篤堅
指導教授-邱瓊萱
關鍵字(中) 醫學專業素養
教育環境
典範
關鍵字(英) medical professionalism
education environment
role model
學科別分類
中文摘要 醫學專業素養為指引醫師、醫學生態度與行為關鍵,故專業素養的構成應為醫學教育的重點之一。本研究之主要目的在於瞭解臺灣醫學專業素養展現情形之現況,以期發現其中問題,進而對醫學專業素養建構環境不足之處提出建議。本研究以五位不同年齡層之醫師與醫學生為研究對象,採質性研究方式,以半結構式訪談來蒐集研究資料,並以開放性編碼方式進行資料整理與分析。分析結果主要分為三大構面,分別為態度建構、個人實行以及環境支持,態度建構此一構面分「共同成長」、「內省」、「平等關係」、「陪伴」、「開放」、「學習苦難」、「關心」以及「利他主義」等八個次構面,其中「共同成長」與「學習苦難」是根據受訪者所說之詞彙為命名依據,其餘七個次構面則是以Cruess等人(2009)之醫治者與專業者的特質為理論依據;個人實行此一構面分為「溝通技巧」、「足夠的專業能力」、「值得信賴的」、「對專業的責任」、「對社會的責任」以及「尊重病人尊嚴與自主性」六個次構面,「溝通技巧」根據文獻回顧結果提出,其他五個次構面也是根據醫治者與專業者的特質而分;環境建構則以受訪者表達的方法為分類依據,共分「支持性的學習環境」、「團隊支持」與「典範學習」三個次構面。本研究之主要結果如下:
一、在態度建構此一構面,世代展現的程度是年長的世代優於年輕的世代,差異的分界在於60~69歲以及50~59歲兩代之間。
二、個人實行之構面則是年輕的世代展現重視程度優於年長的世代,差異的分界則是在50~59歲及40~49歲兩代間。
三、應建構支持性的教學環境,並且建議以團隊支持的機制及發展典範的方式協助學生。
根據研究結果,本研究提出下述建議:一、對於執業經驗豐富之年長世代提倡屬於professional之特質的重要性。二、對於較年輕的世代加強態度建構的部分。三、提升教學環境對於專業素養培育的助力。
英文摘要 Medical professionalism is the attitude and behavior guideline to physicians and medical students. The building of medical professionalism is one of the most important factor in medical education. The purposes of this study are to understand the manifestation of medical professionalism from one generation to another, hoping to see some problems in medical education environment now.
The study is a qualitative design. The study samples consisted three physicians and two medical students. The data were collected by semistructured interviews, transcribed them, and analyzed based on the code and coding. The study result have three aggregate theoretical dimensions: Attitude, practice, and environment support. In attitude, there are eight theoretical categories: grow up together, insight, relations on an equal basis, presence/accompany, openness, learning sufferings, care/compassion, and altruism; In practice, there are six theoretical categories: communication skills, competpence, trustworthy, responsibility to profession, responsibility to society, and respect patient dignity autonomy; In environment support, there are three theoretical categories: supportive learning environment, team support, and role model.
The major findings are:
1. In the attitude aspect, the older generation was better then younger.
2. In the personal behavior aspect, the younger generation was better then older..
3. Supportive environment is necessary for medical education, “team support” and “role model” can help students.

Based on the findings, there are four suggestions: (1) Advocate learning the factors of professional in older generation. (2) Enhance the attitude to medical professionalism in younger generation. (3) Enhance the support in education environment.
論文目次 誌 謝 I
中 文 摘 要 II
Abstract III
目 錄 IV
表目次 V
圖目次 VI
第一章 前言 1
第一節 研究背景 1
第二節 研究目的 3
第二章 文獻探討 4
第一節 醫學專業素養的起源與定義 4
第二節 溝通技巧的定義與相關研究 11
第三節 質性研究與編碼 16
第四節 總結 19
第三章 研究方法與設計 21
第一節 研究架構 22
第二節 研究假設 23
第三節 研究材料與方法 24
第四節 資料處理及分析方法 27
第四章 研究結果 28
第一節 構面整理 28
第二節 構面特質 30
第三節 總結 53
第五章 結論與討論 57
第一節 研究結論與討論 57
第二節 研究限制 62
第三節 研究建議 63
參考文獻 64

參考文獻 中文部份

沈宛甄、柯依萍(2007)。心理辭典。於2010年6月10日,取自:http://coun.nctu.edu.tw/counsel/coun_chin/share/heart_new/200704/dictionary_link.htm
胡幼慧(1996)。轉型中的質性研究:演變、批判和女性主義研究觀點。載於胡幼慧主編,質性研究:理論、方法及本土女性研究實例,頁7-26。台北:巨流圖書。
徐宗國(1996)。紮根理論研究法:淵源、原則、技術與涵義。載於胡幼慧主編,質性研究:理論、方法及本土女性研究實例,頁47-74,台北:巨流圖書。
翁懿涵、許瀛方、黃瓈葳(2000)。紮根理論。於2009年12月20日,取自:http://web.ed.ntnu.edu.tw/~panhu/groundedtheory.pdf
許正眉、蔡佩渝、鄭適芬、李佩怡(2009)。同儕支持團體協助安寧團隊工作者情緒壓力紓解之應用──以彰化基督教醫院為例。安寧療護,14(3),321-330。
張松齡、羅淑芬、王文秀、賴美滿、張秀香、賴彩霞(2003)。東部地區精神科護理人員工作壓力、因應行為及其相關因素之探討。慈濟護理雜誌,2(3),59-69。
黃聖媖、陳怡伶、蔡美慧與曹逢甫(2008)。情境寒暄語與病人回應量之關係:以醫老互動為例。清華學報,38(3),451-484。
葉美玉、康清雲(2003)。醫師衛教門診評價研究—病人觀點。台灣衛誌,22(2),141-146。
葛建培、卓正欽(2008)。績效管理與發展—建構組織人力資本工具與應用。台北:雙葉書廊。
翟曉梅(2009)。論醫患信託關係與醫學的專業精神。臺灣醫學人文學刊,10,135-142。
蔡美慧、盧豐華(2001)。適當的回應病人:從言談技巧改善醫病關係。醫學教育,5(3),61-68。
蔡美慧、曹逢甫、盧豐華(2004)。揭開病人面具—從傾聽病人開始。醫學教育,8(3),96-108。
質性研究資料分析(張芬芬譯)(2005)。臺北市:雙葉書廊。(原著出版年:1994年)

英文部份

ACGME Core Competencies. (n. d.). Retrieved December 10, 2009, from
http://www.mcw.edu/display/docid2478.htm
Arora, N. K., & Gustafson, D. H. (2008). Perceived Helpfulness of Physicians’ Communication Behavior and Breast Cancer Patients’ Level of Trust Over Time. Society of General Internal Medicine, 24(2), 252-255.
Batenturg, V., Smal, F. J., Lodder, A., & Melker, R. A. (1999). Are professional attitudes related to gender and medical specialty? Medical Education, 33, 489-492.
British Medical Association (2001). The Medical Profession and Human Rights: handbook for a changing agenda. London: Zed Books.
Barondess, J. A. (2003). Medicine and Professionalism. ARCH INTERN MED, 163, 145-149.
Brinkmen, W. B., Geraghty, S. R., Lanphear, B. P., Khoury, J. C., Rey, J. A. G., & DeWitt, T. G. et al. (2006). Evaluation of Resident Communication Skills and Professionalism: A Matter of Perspective? American Academy of Pediatrics, 118(4), 1371-1379.
Brinkmen, W. B., Geraghty, S. R., Lanphear, B. P., Khoury, J. C., Rey, J. A. G., & DeWitt, T. G. et al. (2007). Effect of Multisource Feedback on Resident Communication Skills and Professionalism. ARCH PEDIATR ADOLESC MED, 161, 44-49.
Cruess, R., Sylvia, R., & Cruess, S. (2009). Teaching Medical Professionalism. NY: Cambridge University Press.
Chiu, C. H., Arrigo, L. G., & Tsai, D. (2009). History Contest for the Growth of Medical Professionalism and Curriculum Reform in Taiwan. Kaohsiung J Med Sci, 25(9), 510-514.
Chun, M. B. J., Young, K. G. M., & Jack, D. S. (2009). Incorporating cultural competency into the general surgery residency curriculum: A preliminary assessment. International Journal of Surgery, 7, 368-372.
Diaz, J. A., & Stamp, M. J. (2004). Journal of the American Podiatric Medical Association, 94(2), 206-209.
Dobie, S. (2007). Viewpoint: Reflection on a Well-Traveled Path: Self-Awareness, Mindful Practice, and Relationship-Centered Care as Foundations for Medical Education. Academic Medicine, 82(4), 422-427.
Francis, C. K. (2002). Professionalism and the medical student. Ann Intern Med, 136, 1647.
Hafferty, F. W. (2002). What Medical Students Know about Professionalism. THE MOUNT SINAI JOURNAL OF MEDICINE, 69(6), 385-397.
Hojat, M., Vergare, M. J., Maxwell, K., Brainard, G., Herrine, S. K., & Isenberg, G. A. et al. (2009). The devil is in the third year: A longitudinal study of erosion of empathy in medical school. Academic Medicine, 84(9), 1182-1191.
Johansson, E. E., & Hamberg, K. (2007). From calling to a scheduled vocation: Swedish male and female students’ reflections on being a doctor. Medical Teacher, 29, e1-e8.
Killip, S., Carol, L., Ireson, R. N., Margaret, M. L., Steven, Fleming, T., & Katirai, W. et al. (2007). Patient Safety in After-hours Telephone Medicine. Family Medicine, 39(6), 404-409.
Mueller, P. S., Barrier, P. A., Call, T. G., Duncan, A. K., Hurley, D. L., & Multari, A. et al. (2006). Views of new internal medicine faculty of their preparedness and competence in physician-patient communication. BMC Medical Education, 6(30), 1472-1479.
Mast, M. S., Hall, J. A., Klockner, C., & Choi, E. (2008). Medical Care, 46(12), 1212-1218.
Ong, L. M. L., Haes, J. C. J. M., Hoos, A. M., & Lammes, F. B. (1995). Doctor- patient communication: A review of the literature. Social Science and Medicine, 40(7), 903-918.
Reisman, A. B., & Brown, K. E. (2005). Preventing Communication Errors in Telephone Medicine: A Case-Based Approach. J GEN INTERN MED, 20, 959-963.
Sullivan, W. M. (2000). Medicine under threat: Professionalism and professional identity. CMAJ, 162(5), 673-675.
Stevens, R. A. (2001). Public roles for the medical profession in the United States: Beyond theories of decline and fall. The Milbank Quarterly, 79(3), 327-353.
Tsugawa, Y., Tokuda, Y., Ohbu, S., Okubo, T., Cruess, R., & Cruess, S. (2009). Professionalism Mini-Evaluation Exercise for medical resident in Japan: A pilot study. Medical Education, 43, 968-978.
Veloski, J. J., Fields, S. K., Boex, J. R., & Blank, L. L. (2005). Measuring professionalism: A review of studies with instruments reported in the literature between 1982 and 2002. Academic Medicine, 80(4), 366-370.
Whitson, H. E., Hastings, S. N., Eleanor, McConnell, E. S.,& Lekan-Rutledge. (2006). Inter-disciplinary Focus Groups on Telephone Medicine: A Quality Improvement Initiative. American Medical Directors Association, 7, 407-411.
Weng, H. C., Chen, H. C., Chen, H. J., Lu, K., & Hung, S. Y. (2008). Doctors’ emotional intelligence and the patient-doctor relationship. Medical Education, 42, 703–711.

------------------------------------------------------------------------ 第 19 筆 ---------------------------------------------------------------------
系統識別號 U0007-0707201012584000
論文名稱(中文) 壓抑, 解脫與認同-以鄭清文五篇短篇小說的敘事分析為例
校院名稱 臺北醫學大學
系所名稱(中) 醫學人文研究所
系所名稱(英) Graduate Institute of Humanities in Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 張簡士暄
學號 M116097001
學位類別 碩士
語文別 中文
口試日期 2010-05-13
論文頁數 110頁
口試委員 指導教授-丁興祥
共同指導教授-陳永興
委員-周慶塘
委員-王恩南
委員-何信翰
關鍵字(中) 鄭清文
敘事分析
防衛機轉
文本分析
自我認同
關鍵字(英) Qing-wen Zheng
Narrative Analysis
Defense Mechanisms
Story Analysis
Self-identity
學科別分類
中文摘要 現今以「文本」作為研究、分析的題材已相當普遍,它們試圖從不同的理論觀點出發和切入,無論是從社會、歷史、文化、人類、心理學等不同視野皆已累積相當豐富的成果,當中特別是用心理學的角度、概念來分析、探討文本更可以直接來說明故事人物與情境間互動的心理歷程和因應模式進而去投射小說創作年代背景下,社會所呈現的特殊心理現象與性格。受到世界潮流的影響,九零年代當文化(本土)心理學崛起時,有越來越多心理學者開始關注自身的生活經驗與所生長的土地,丁興祥、宋文里、余德慧等國內知名心理學者開始運用社會、文化脈絡來研究人心理的現象,也間接使國內心理學研究更趨多樣化。

本文以臺灣重要鄉土文學作家鄭清文的短篇小說為研究題材,儘管在本文鎖定的上百篇小說中各自有不一樣的主題與涵義,但在反覆閱讀之中不難發現卑微的人物角色皆在困頓環境中尋找、追求解放的歷程,而主要的目的是透過以心理學的觀點、概念分析台灣文學小說來更關注台灣當代的社會。

本文是以Catherine Kohler Riessman的敘事分析(Narrative Analysis)作為研究的方法,而主要的分析概念是來自於精神分析理論,分析的過程分三個層次,在第一層分析裡,藉由精神分析中所提到的防衛機轉、死和生的本能、精神動能(psycho-dynamic)、人格結構四項概念為基礎,透過上下文、小說人物的對話、和整體故事氣氛來看其人物的心理現象;第二層次的分析中同樣是以敍事分析為背景的美國心理學家Gergen的敘事形式(Narrative Form)試圖從整體故事找到故事的主調(tone),並觀察人物主角是否達成目標;第三層次分析本文試圖找到故事中的關鍵性故事發生事件並與當代社會背景作連結來間接說明、隱射與解釋社會現象。

在閱讀上百篇作品所歸納出五篇具有相同的時代背景,顯著的心理衝突現象的小說:<雞>、<我的「傑作」>、<水上組曲>、<秘密>、<皇帝魚的二次災厄>之後,這五篇所要傳達的共同主題是小說人物所呈現「壓抑」的心理狀態,並在討論部份經由Erikson核心理論-認同(Identity)來進一步做延伸探討與解釋個體在壓抑心理的困頓中如何尋找自己的定位與在自我認同過程中是否扮演、調適好自己的角色,並朝著自己認為理想的路途、目標前進。
論文目次 目次
第一章 緒論...............................................1
第一節 研究動機與目的...........................................1
第二章 文獻回顧...........................................9
第一節 佛洛伊德在1926年的手稿..................................9
第二節 Erik H. Erikson在青年路德身上看見的認同危機.............15
第三節 運用精神分析來進行文本分析的案例........................18
第四節 運用敍事分析研究個案的相關文獻..........................20
第三章 研究方法..........................................24
第一節 研究取向:敍事分析......................................24
第二節 How to Analysis?........................................28
第四章 研究結果..........................................30
故事一 水上組曲...............................................30
故事二 我的「傑作」...........................................44
故事三 雞.....................................................56
故事四 秘密...................................................71
故事五 皇帝魚的二次災厄.......................................84
第五章 結論與討論........................................95
第一節 壓抑與解脫.............................................95
第二節 文化生活投射於小說....................................100
第三節 契合性................................................103
第四節 研究限制、建議.........................................104
第五節 有效性(評鑑指標).....................................106
第六章 參考文獻.........................................109
參考文獻 中文部份
丁興祥、張慈宜、曾寶瑩(譯)(2006),Smith.J.A(2003):質性心理學研究法的實務指南,台北:遠流出版社。

宋文里主編(2007)。《應用心理研究》,第34期,第43頁。台北:五南出版社。

李喬、葉石濤、彭瑞金(1982)。鄭清文作品討論會,文學界,二期。

汪暉(1988)。作為哲學人類學的佛洛伊德理論,台北:遠流出版社。

林瑞明主編(1993)。以生命的熱情觀察人生-鄭清文集70年代洪醒夫對鄭清文 的訪談,第9~14頁。台北:前衛出版社。

康綠島(譯)(1989),Erikson E.H(1958):青年路德,台北:遠流出版社。

張美玲(2008)。《鄭清文小說死亡書寫研究》。台北:國立台北教育大學碩士論文。

鄭谷苑著(2007)。走出峽地:鄭清文的人生故事。台北:麥田、城邦文化。

鄧明宇、王勇智(譯)(2003),丁興祥(校訂)(2003),Riessman, C.K.(1993):《敘說分析》,台北:五南出版社。


英文部份
Lysaker Paul Henry, Lysaker John Timothy (2002). “Narrative Structure in Psychosis: Schizophrenia and Disruptions in the Dialogical Self”, Social Science Collections. Vol. 12(2): 207~220.

Riessman, Catherine Kohler (2002), “Narrative Analysis.”. Thousand Oaks, CA.:Sage.

Roe D, Davidson L (2005). “Self and narrative in schizophrenia: time to author a new story”, Medical Humanities, 31: 89~94.

Sigmund Freud, translated from the German under the General Editorship of James Strachey in collaboration with Anna Freud assisted by Alix Strachey and Alan Tyson(1995).The Standard edition of the complete psychological works Volume XX(1925~1926),” Inhibitions, Symptoms, and Anxiety”, Published by The Hogarth Press Limited, Clarke, Irwin and Co. Ltd, Toronto.

Theodore R. Sarbin, Gergen, K.J.and Gergen, M. M.(1986)Narrative Form and the Construction of Psychological Science ,Sarbin, T.R.(Ed)(1986)Praeger Special Studies, Praeger Scientific, New York, Westport. Conneticut, London.





 


若您有任何疑問,請與我們聯絡!
臺北醫學大學 圖書館 簡莉婷
E-mail:etds@tmu.edu.tw
Tel:(02) 2736-1661 ext.2519
Fax:(02) 2737-5446