系統識別號 U0007-1302200815104900
論文名稱(中文) 備勞喘吸入劑抑制脂多醣刺激巨噬細胞產生間白素-六基因分子機制研究
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 96
學期 1
出版年 97
研究生(中文) 張哲華
研究生(英文) Jer-Hwa Chang
學號 M102094030
學位類別 碩士
語文別 英文
口試日期 2008-01-17
論文頁數 47頁
口試委員 指導教授-陳大樑
中文關鍵字 備勞喘  脂多醣  巨噬細胞  間白素-六 
中文摘要 備勞喘(fenoterol)吸入劑為一種短效且具選擇性之乙二型交感神經刺激劑(beta2-agonist),常被廣泛地使用於阻塞性肺部疾病或是嚴重肺部感染症而導致急性呼吸窘迫症(ARDS)之病患,作為控制其支氣管攣縮之藥物。引起急性呼吸窘迫症(ARDS)最普遍的原因為嚴重感染,而某些乙型交感神經刺激劑,經近期研究發現可被用於早期緩解急性呼吸窘迫症。備勞喘在過去的研究中極少被探討關於抗發炎之效果,對於其作用機制亦未曾被闡明。一些先發研究顯示,在動物或人體實驗中,乙型交感神經刺激劑可抑制細胞激素,例如:腫瘤壞死因子(TNF)以及間白素-六(IL-6)之生成。本研究之目的在評估備勞喘在經脂多醣(LPS)活化之RAW264.7類巨噬細胞模式中,是否可以產生IL-6 mRNA之免疫抑制效應,並且探討其可能之分子機制。
RAW264.7巨噬細胞於不同濃度1, 10及100 x 10-3 M之備勞喘劑下,經有或無100 ng/ml LPS加入,在一、六及二十四小時之時間間隔後,皆對細胞存活率(viability)沒有影響。 以備勞喘及LPS處理RAW264.7,LPS可促使IL-6 mRNA之產生,並在第一及第六小時被備勞喘所抑制。為了探討備勞喘抑制IL-6 mRNA之分子機制,我們研究不同的轉錄因子。在LPS活化之巨噬細胞中,備勞喘能於兩個小時內顯著地抑制c-Jun核蛋白質。在被LPS活化之巨噬細胞中,備勞喘也能於一個小時內顯著地抑制c-Jun總細胞蛋白,但無法抑制c-Fos或NF-?羠蛋白。為了闡明c-Jun的上游路徑,我們也設計研究p-JNK以及p-MEK-4蛋白。結果顯示,在兩個小時內被LPS活化之RAW264.7中,p-JNK 1/2蛋白,可被備勞喘不顯著地抑制。在活化之RAW264.7中且於五十分鐘內,經備勞喘處理後,p-MEK-4蛋白亦可被備勞喘不顯著地抑制。
本研究結果顯示備勞喘可抑制IL-6 mRNA,且其機轉可能是透過c-Jun或許也可能是透過與JNK/MEK-4有關之分子路徑。
英文摘要 Fenoterol inhalant solution, a short-acting and selective beta2-agonist, is widely used as aerosal inhalation for management of bronchospasm in patients with obstructive lung disease or severe lung infection. The most common cause of acute respiratory distress syndrome (ARDS) is severe infection, and some beta-adrenergic agents are under studies as novel treatments targeting earlier resolution of ARDS. The studies of fenoterol for the anti-inflammation effect are rare and no known mechanism has been elucidated in previous articles. Previous studies revealed that beta-agonist caused cytokine inhibition (e.g. TNF and IL-6 production) in animal or human models. The aims of this studies were to evaluate the effect of fenoterol on IL-6 mRNA production and its possible molecular mechanisms in the cell model of lipopolysaccharide (LPS)-activated macrophage-like cell line, RAW264.7.
Exposure of RAW264.7 macrophages to 1, 10, 100 x 10-3 M fenoterol at 1-, 6-, and 24-hour intervals with or without 100 ng/ml LPS did not affect cell viability. While treated with fenoterol and LPS in RAW264.7, the IL-6 mRNA production was induced by LPS and inhibited at the 1st and the 6th hours. To clarify the molecular mechanisms by which fenoterol inhibited the IL-6 mRNA, different transcription factors were investigated. Fenoterol could significantly suppress the nuclear protein levels of c-Jun in LPS-activated macrophages within 2 hours. Fenoterol could also significantly suppress the total cellular protein levels of c-Jun but not c-Fos or NF-?羠 in LPS-activated macrophages within 1 hour. In order to elucidate the upstream pathways, the p-JNK (phosphorylated c-Jun N-terminal kinase) and p-MEK-4 (phosphorylated MAP kinase kinase 4) proteins were studied. The results revealed that p-JNK1/2 proteins were insignificantly suppressed by fenoterol within 2 hours after the LPS activation in RAW264.7. The p-MEK-4 proteins levels were also insignificantly suppressed by fenoterol within 50 minutes in LPS activated macrophages.
The results of this study showed that fenoterol could suppress IL-6 mRNA expression through c-Jun and possibly JNK/MEK-4-dependent pathway.
論文目次 Contents

Index of Tables I
Index of Figures II
Abstract in Chinese III
Abstract V
Introduction 1
I. Lung infection and alveolar macrophages 1
II. Beta 2-agonists in acute lung injury 3
III. Immunosuppression of beta agonists and fenoterol 4
IV. Lipopolysaccharides, inflammatory cytokines and lung injuries 7
V. MAPK cascades in the signaling pathways 8
VI. MAPK and inflammatory cytokines 9
VII. C-Jun nuclear protein regulation 10
VIII. Study hypothesis and specific aims 10
IX. Study design 12
Materials and Methods 14
I. Experimental materials 14
II. Cell culture and drug treatment 14
III. Determination of cell viability 15
IV. Reverse-transcription polymerase chain reaction (RT-PCR) assay 15
V. Preparation of nuclear and total cell proteins and immunodetection analyses 16
VI. Immunodetection of JNK/MEK-4 and phosphorylated JNK/MEK-4 17
VII. Statistical analysis 17
Results 19
I. Effects of fenoterol and LPS on cell viability of macrophages 19
II. Effects of fenoterol on IL-6 mRNA production in LPS-activated macrophages 19
III. Effects of fenoterol on nuclear and total protein c-Jun production in LPS-activated macrophages 20
IV. Effects of fenoterol on other total proteins (NF-?羠 and c-Fos) production in LPS-activated macrophages 21
V. Effects of fenoterol on p-JNK proteins production in LPS-activated macrophages 22

VI. Effects of fenoterol on p-MEK4 proteins production in LPS-activated macrophages 23
Discussion 24
I. Effects of fenoterol and LPS on cell viability of macrophages 25
II. Effects of fenoterol on IL-6 mRNA production in LPS-activated macrophages 26
III. Effects of fenoterol on nuclear and total proteins (c-Jun, NF-?羠 and c-Fos) production in LPS-activated macrophages 26
IV. Effects of fenoterol on p-JNK proteins production in LPS-activated macrophages 27
V. Effects of fenoterol on p-MEK4 proteins production in LPS-activated macrophages 28
Conclusion 29
References 30
Tables and Figures 37
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