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系統識別號 U0026-1408201212215100
論文名稱(中文) 菸萃取物誘導前胸腺素過量表現產生肺氣腫
論文名稱(英文) Overexpression of prothymosin α induced by cigarette smoke extract generates pulmonary emphysema
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 相雅潔
研究生(英文) Ya-Chieh Shiang
學號 s16994077
學位類別 碩士
語文別 中文
論文頁數 40頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-賴明德
口試委員-曾堯麟
口試委員-李國榮
中文關鍵字 前胸腺素  肺氣腫  菸萃取液 
英文關鍵字 prothymosin α  emphysema  cigarette smoke extract 
學科別分類
中文摘要 肺氣腫 (Emphysema) 是主要的一種慢性阻塞性肺疾病 (Chronic Obstructive Pulmonary Disease, COPD) 的典型,病徵為肺泡壁的瓦解以及肺泡氣室的異常擴張,導致肺臟失去彈性,呼吸系統失能。根據世界衛生組織估計,慢性阻塞性肺疾病將於 2020 年成為全球人類的第三大死因,對人類健康的影響甚鉅。引起肺氣腫的主因為長期性的抽菸,然而卻僅有 20% 的抽菸族群成為肺氣腫患者,其中顯然有未定義的因子參與。前胸腺素 (Prothymosin α, 簡稱 ProT) 在免疫與氧化壓力的調節扮演重要角色,在過去實驗室的研究發現 ProT 過量表現的同型合子小鼠會自發性的產生肺氣腫,並且在肺氣腫患者的組織切片中看到 ProT 有過量表現。因此,本研究主要探討 ProT 與香菸引起的肺氣腫模式之間的關聯。我們發現給予菸萃取液的大鼠在肺氣腫區域會大量表現 ProT 及磷酸化 NF-κB。在 ProT 同型合子小鼠的肺氣腫模式中也發現磷酸化 NF-κB 及基質金屬蛋白酵素-12 (Matrix metalloproteinase-12, 簡稱 MMP-12) 會過量表現。因此,我們利用 ProT 基因轉殖鼠做香菸誘導肺氣腫模式的研究,發現 ProT 異型合子小鼠較一般小鼠嚴重,且磷酸化 NF-κB 及 MMP-12 也會過量表現。若抑制小鼠肺部 ProT 的表現能有效預防香菸造成的肺氣腫模式,且磷酸化 NF-κB 及 MMP-12 的表現量皆下降。更進一步,若是外給 ProT 基因到 MMP-12 基因剃除鼠的肺臟中表現,則能保護小鼠免於肺氣腫生成。我們也在細胞實驗上證實菸萃取液會造成 ProT 及磷酸化 NF-κB 的表現量增加。綜合以上結果,本篇研究指出 ProT 是藉由促進 NF-κB 磷酸化進而使 MMP-12 的表現量增加,在香菸誘導的肺氣腫模式中扮演重要角色。
英文摘要 Pulmonary emphysema, a major subtype of chronic obstructive pulmonary disease (COPD) predicted to become the third most common cause of death worldwide by 2020, is defined as the enlargement of alveolar airspace and the loss of lung elasticity. This disease is mostly caused by cigarette smoking. Despite being the main risk factor, only about 20% of cigarette smokers develop emphysema. There may be unidentified genes predisposing to cigarette smoke-induced emphysema. Prothymosin α (ProT) is an acidic nuclear protein associated with oxidative stress and immunomodulation. In our previous studies, we demonstrated that ProT homozygous transgenic mice exhibit a emphysema-like phenotype. We also detected ProT overexpression in lung epithelium from smokers with emphysema. In this study, we investigated whether ProT is involved in the pathogenesis of cigarette smoke extract (CSE)-induced emphysema. In the rat model, we showed that ProT and phosphorylated NF-κB were overexpressed in the emphysematous parts after induction with CSE. Phosphorylated NF-κB and MMP-12 were also overexpressed in ProT homozygous transgenic mice with emphysema. Furthermore, ProT heterozygous mice treated with CSE developed more severe emphysema as compared to CSE-treated wild-type mice. Phosphorylated NF-κB and MMP-12 were overexpressed in the ProT transgenic mice after treatment with CSE. By contrast, CSE-induced emphysema could be prevented by inhibition of ProT expression. Phosphorylated NF-κB and MMP-12 were downregulated in the ProT knockdown mice. In addition, ProT-induced emphysema could also be prevented in MMP-12 knockout mice. Our in vitro study also showed that expressions of ProT and phosphorylated NF-κB were upregulated in A549 cells after treatment with CSE. Taken together, our studies demonstrate that ProT plays an important role in CSE-induced emphysema through upregulation of phosphorylated NF-κB and MMP-12.
論文目次 考試合格證明 I
中文摘要 Ⅱ
Abstract Ⅳ
誌謝 Ⅵ
總目錄 Ⅶ
圖目錄 X
縮寫 XI
第一章 緒論 1
一、肺氣腫 (Emphysema) 與吸菸 1
二、肺氣腫之動物模式與基質金屬蛋白酵素 (MMP) 3
三、前胸腺素 (Prothymosin α, ProT) 4
第二章 研究目的 6
第三章 材料與方法 7
一、材料 7
1、菸萃取液 7
2、實驗動物 7
(1)S.D. 大鼠 7
(2)FvB 小鼠 7
(3)ProT 異型合子小鼠 7
(4)MMP-12 基因剃除鼠 7
3、細胞株 8
4、引子 8
5、質體 8
6、試劑 9
7、勝任細胞 11
8、抗體 11
二、方法 13
1、菸萃取液誘導肺氣腫之動物模式 13
(1)大鼠動物模式 13
(2)小鼠動物模式 13
(3)細胞實驗 13
2、免疫組織化學染色法 (IHC) 14
3、肺泡擴大的百分比 14
4、肺氣腫發病率 15
5、慢病毒生產 15
6、慢病毒濃縮 16
7、氣管給予慢病毒 16
8、細胞培養 17
9、RNA 萃取 17
10、反轉錄酶聚合連鎖反應 (RT-PCR) 18
11、即時半反應反轉錄酶聚合連鎖反應 (Real-time PCR) 18
12、西方墨點法 (Western blot) 19
第四章 結果 20
一、前胸腺素 (ProT) 在菸萃取液誘導的肺氣腫區域有大量表現 20
二、ProT 基因轉殖小鼠對於菸誘導的肺氣腫模式具較高感受性 20
三、抑制小鼠肺部 ProT 的表現對菸誘導的肺氣腫有預防效果 21
四、ProT 過量表現的同型合子小鼠會自發性產生肺氣腫,且磷酸
化NF-κB 及 MMP-12 會過量表現 21
五、MMP-12 基因剃除鼠能預防 ProT 過量表現造成的肺氣腫 22
六、A549 細胞株在菸萃取液的刺激下會大量表現 ProT 及磷酸化NF-κB 22
第五章 結論 24
第六章 討論 25
參考文獻 28
圖表 32

圖目錄
圖一、 菸萃取液 (CSE) 成功誘導大鼠產生肺氣腫 32
圖二、 ProT 及磷酸化 NF-κB 在菸誘導的肺氣腫區域有大量表現 33
圖三、 ProT 基因轉殖小鼠在菸誘導的肺氣腫中較為嚴重 34
圖四、 ProT 基因轉殖小鼠在給予菸萃取液後會大量表現 ProT、磷酸
化 NF-κB 及 MMP-12 35
圖五、 抑制小鼠肺部 ProT 表現對於菸誘導的肺氣腫有預防效果 36
圖六、 抑制小鼠肺部 ProT 表現的同時給菸萃取液,ProT 及 MMP-12
的表現量無法受菸萃取液的刺激而上升 37
圖七、 ProT 同型合子小鼠會過量表現磷酸化 NF-κB 及 MMP-12 38
圖八、 MMP-12 基因剃除鼠能預防 ProT 過量表現造成的肺氣腫 39
圖九、 A549 細胞在菸的處理下,ProT 及磷酸化 NF-κB 表現增加有
相同趨勢;若抑制小鼠肺部 ProT 表現,則 MMP-12 也受抑制 40
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