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系統識別號 U0026-0608201311180200
論文名稱(中文) 探討前胸腺素在肺纖維化致病機轉的角色
論文名稱(英文) The Role of prothymosin α in the pathogenesis of pulmonary fibrosis
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 101
學期 2
出版年 102
研究生(中文) 莊舒涵
研究生(英文) Shu-Han Chuang
學號 s16004115
學位類別 碩士
語文別 中文
論文頁數 54頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-曾堯麟
口試委員-蔣思澈
口試委員-李哲欣
中文關鍵字 肺纖維化  乙型轉化生長因子  上皮-間質轉化  前胸腺素 
英文關鍵字 pulmonary fibrosis  TGF-β  epithelial-mesenchymal transition  EMT  prothymosin α 
學科別分類
中文摘要 肺上皮細胞重複性受到損傷可能會因為許多肺泡的微環境的改變,促進肺上皮細胞減少以及間質性細胞堆積,導致不正常的細胞修復以及重建,肺間質的組織增厚,造成肺組織喪失氧氣交換的能力,呼吸系統失能,且過多的細胞外基質(Extracellular matrix, ECM)堆積在肺中導致肺纖維化(Pulmonary fibrosis)。促進纖維化因子是影響肺泡微環境之一,其中乙型轉化生長因子(Transforming growth factor-β, TGF-β)與組織發育、致癌以及纖維化有關,也是上皮-間質轉化(Epithelial-mesenchymal transition, EMT)的主要誘導者。EMT是上皮細胞轉變成間質型細胞的過程,在肺損傷後的組織修復與傷疤的形成扮演重要的角色。前胸腺素(Prothymosin α, ProT)是酸性核蛋白,會調節細胞的轉錄、染色質重建以及免疫調節等,在過去實驗室的研究發現在四氯化碳(CCl4)誘導的肝纖維化中,ProT可以調控Smad7 蛋白的表現。因此,我們假設ProT可能通過抑制TGF-β訊息傳遞路徑來抑制肺纖維化,利用博萊黴素(Bleomycin)誘導肺纖維化的動物模式中研究ProT在肺損傷以及纖維化中的致病機轉。我們在細胞實驗上證實在TGF-β誘導後,ProT過量表現的肺上皮細胞中,磷酸化Smad2表現會下降,且被誘導增加的Snail及N-cadherin也因為ProT的過量表現而被抑制了,相反的E-cadherin回升,也發現ProT會抑制細胞爬行的
能力,另外在ProT下降表現的細胞中有一致的證實。在動物實驗方面,首先我們發現在ProT基因轉殖鼠的肺臟中,Smad7的表現量較高。而在小鼠肺纖維化的模式中,發現ProT基因轉殖鼠磷酸化Smad2及纖維母細胞特異性蛋白(Fibroblast specific protein 1, FSP1)的表現量減少,另一方面也發現肺纖維化在一般小鼠較ProT基因轉殖鼠嚴重。綜合以上結果,本篇研究指出ProT藉由增加Smad7的表現來抑制TGF-β誘導的EMT中扮演重要角色,並且可以減少肺纖維化的發生。
英文摘要 Pulmonary fibrosis is the formation or development of excess deposition of extracellular matrix in the lung. Repetitive injury of alveolar epithelium may cause dysregulated repair and aberrant tissue remodeling. Many alterations in the alveolar microenvironment eventually promote loss of alveolar epithelial cells and accumulation of activated fibroblasts and myofibroblasts, leading to progressive fibrosis. Transforming growth factor-β (TGF-β) is one of the profibrogenic cytokines responsible for tissue development, carcinogenesis, and fibrosis. It is also a major inducer of epithelial-mesenchymal transition (EMT). EMT is a process required for epithelial cells undergoing phenotypic changes to mesenchymal counterparts and gives rise to myofibroblasts, which plays an important role in tissue repair and scar formation following epithelial injury. Prothymosin α (ProT) is an acidic nuclear protein and an important regulator of cell proliferation, transcription, chromatin remodeling, and immunomodulation. We have demonstrated previously that ProT can upregulate smad7 protein levels and reduce CCl4-induced liver fibrosis in mice. Therefore, we hypothesized that ProT may play a pivotal role in suppressing pulmonary fibrosis through the inhibition of TGF-β signaling pathway. Here we studied the role of ProT in the pathogenesis of lung injury and fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis. We showed that phosphorylated smad2 levels were reduced in ProT-overexpressing lung epithelial cells after treatment with TGF-β. We also found that TGF-β-induced downregulation of E-cadherin and upregulation of Snail and N-cadherin could be suppressed by ProT overexpression in A549 lung epithelium-like cells. Furthermore, ProT transgenic mice expressed higher levels of smad7 in the lung. Therefore in our animal model, we found that pulmonary fibrosis and fibroblast specific protein 1 (FSP1) were reduced in ProT transgenic mice compared to non-transgenic mice after bleomycin treatments. Taken together, our results suggest an important regulatory role of ProT in the inhibition of TGF-β-induced EMT through the upregulation of smad7.
論文目次 總目錄
考試合格證明 I
中文摘要 Ⅱ
Abstract Ⅳ
誌謝 Ⅵ
總目錄 Ⅷ
圖目錄 XIII
縮寫 XⅣ
第一章 緒論 1
一、肺纖維化 (Pulmonary fibrosis) 1
二、乙型轉化生長因子 (Transforming growth factor-β, TGF-β) 2
三、上皮-間質轉化 (Epithelial-mesenchymal transition, EMT) 3
四、乙型轉化生長因子訊息傳遞路徑 (TGF-β signaling pathway) 3
五、前胸腺素 (Prothymosin α, ProT) 4
第二章 研究動機與目的 6
第三章 材料與方法 8
一、實驗材料 8
1.質體 8
1-1.表現載體 8
1-2.RNA干擾質體 8
2.細胞株 9
3.引子 9
4.抗體 10
4-1.一級抗體 10
4-2.二級抗體 11
4-3.ELISA Kit 11
5.實驗動物 11
6.菌株 11
7.試劑 12
7-1.細菌培養液 12
7-2.細胞處理緩衝液 12
7-3.緩衝液 13
8.藥物 14
9.重組蛋白 14
二、實驗方法 15
1.細胞培養 15
1-1.HEK 293T細胞、A549細胞 15
1-2.MLE-12細胞 15
2.慢病毒生產與感染 16
2-1.慢病毒生產 16
2-2.慢病毒感染與加藥篩選 16
3.西方墨點法 17
3-1.蛋白樣品製備 17
3-2.蛋白電泳與免疫轉漬 17
4.RNA萃取 18
5.反轉錄 18
6.聚合酶連鎖反應 19
7.Boyden chamber assay 20
8.動物實驗 20
8-1.身分確認 20
8-1-1.取得小鼠染色體DNA 20
8-1-2.PCR確認基因型-ProT轉殖基因小鼠 21
8-2.肺纖維化動物模式氣管給予Bleomycin 21
8-3.組織固定與包埋 22
8-4.動物檢體切片染色法 22
8-4-1.H&E染色 22
8-4-2.Masson’s trichrome組織學染色 22
8-4-3.免疫組織化學染色法 23
8-5.動物檢體切片染色後之評分方式 24
8-5-1.H&E評分方式 24
8-5-2.Masson’s trichrome stain定量方式 24
8-5-3.IHC評分方式 24
9.酵素免疫分析法 24
10.統計分析 25
第四章 結果 26
一、Smad7大量表現在ProT基因轉殖小鼠肺臟中 26
二、ProT基因轉殖小鼠對於Bleomycin誘導的肺纖維化模式具較低感受性 26
1.成功建立bleomycin誘導的肺纖維化模式 26
2.正常小鼠的肺纖維化程度較為嚴重 27
3.ProT可抑制TGF-β4. ProT會抑制纖維母細胞的產生 27
4.訊息傳遞路徑在肺纖維化模式中 28
三、肺上皮細胞在ProT過量表現下會抑制TGF-β訊息傳遞路徑 28
1.確認成功建立ProT過量表現以及低表現的細胞株 28
2.在細胞實驗中ProT可抑制TGF-β訊息傳遞路徑 28
四、肺上皮細胞在ProT過量表現下會抑制細胞EMT及爬行能力 29
1.ProT抑制細胞EMT的能力 29
2.ProT抑制細胞爬行的能力 29
第五章 結論 30
第六章 討論 31
參考文獻 36
圖表 43
自述 54

圖目錄
圖一、Smad7在ProT基因轉殖小鼠的肺中有大量表現 43
圖二、成功建立Bleomycin誘導的肺纖維化模式 44
圖三、Bleomycin誘導肺纖維化模式中,ProT基因轉殖小鼠產生肺纖維化較輕微 45
圖四、Bleomycin誘導肺纖維化模式中,ProT基因轉殖小鼠肺臟產生的膠原蛋白沉積較正常小鼠少 46
圖五、Bleomycin誘導肺纖維化模式中,ProT基因轉殖小鼠肺臟的Smad7有大量表現並會抑制TGF-β-Smad訊息傳遞路徑 48
圖六、Bleomycin誘導肺纖維化模式中,ProT基因轉殖小鼠肺臟的FSP1表現量下降 49
圖七、ProT過量表現的細胞株中,磷酸化Smad2表現量被抑制 50
圖八、ProT抑制表現的細胞株中,磷酸化Smad2會大量表現 51
圖九、ProT會抑制肺上皮細胞A549上皮-間質轉化的能力 52
圖十、ProT會抑制肺上皮細胞A549細胞爬行的能力 53
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