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研究生:陳沛瀠
研究生(外文):Pei-Ying Chen
論文名稱:5,7,3',4'-四甲氧基黃酮抑制轉化生長因子β誘導特發性肺纖維化之機制
論文名稱(外文):The mechanism of 5,7,3',4'-tetramethoxyflavone inhibiting TGF-β-induced idiopathic pulmonary fibrosis
指導教授:林季千
指導教授(外文):Chi-Chien Lin
口試委員:童鈺棠林民昆
口試委員(外文):Yu-Tang TungMing-Kuem Lin
口試日期:2023-12-07
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2023
畢業學年度:112
語文別:中文
論文頁數:55
中文關鍵詞:特發性肺纖維化多甲氧基黃酮573'4'-四甲氧基黃酮轉化生長因子β博來黴素
外文關鍵詞:Idiopathic pulmonary fibrosisPolymethoxyflavones573'4'-tetramethoxyflavoneTGF-β signaling pathwayBLM
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特發性肺纖維化 (Idiopathic pulmonary fibrosis, IPF) 是一種慢性且逐漸惡化的肺間質性疾病,臨床上發現患者預後較差。目前治療肺纖維化的藥物有限,無法延長患者的存活期。因此,開發新的治療方法對於肺纖維化的研究至關重要。肺纖維化的病理特徵包括肺部上皮細胞損傷、發炎、肌成纖維細胞形成以及細胞外基質的累積。其中,成纖維細胞在纖維化過程中扮演著重要的角色,並受到轉化生長因子β (TGF-β) 的調控。TGF-β的存在會抑制纖維母細胞的細胞凋亡,導致異常的細胞增生和細胞外基質的產生。因此,減緩TGF-β的促纖維化作用對於防止肺纖維化的惡化至關重要。TGF-β透過誘導上皮間質轉化 (Epithelial-mesenchymal transition, EMT) 加速肺纖維化的發展。EMT是一種複雜的過程,其中上皮細胞失去上皮特性並獲得間質細胞特徵。在肺纖維化中,EMT的特徵是上皮細胞標記的減少,而間質細胞標記的增加。多甲氧基黃酮 (Polymethoxyflavones, PMF) 是柑橘皮中發現的天然化合物,被指出具有較高的生物活性,同時具有抗發炎與抗腫瘤之特性。5,7,3',4'-四甲氧基黃酮(5,7,3',4'-Tetramethoxyflavone, TMF) 是月橘 (Murraya paniculata) 與黑薑 (Kaempferia parviflora) 中主要的PMFs之一。本研究主要探討TMF抑制轉化生長因子β誘導特發性肺纖維化之機制分別建立體外 (in vitro) 與體內 (in vivo) 模型模擬疾病模式。結果顯示,TGF-β (5 ng/mL) 刺激小鼠成纖維細胞NIH/3T3後,會促使細胞增殖 (Proliferation) 與間質細胞的標記物 (α-SMA、CollagenⅠ及Fibronectin) 表現增加,EMT 相關轉錄因子 (Snail) 也被刺激上調。TGF-β的刺激也導致Smad、non-Smad與Wnt/β-catenin 表現顯著增加。我們發現TMF可以抑制Smad 轉錄因子Snail的mRNA表現量,而Smad、non-Smad與Wnt/β-catenin 的表現量透過西方墨點法偵測到TMF處理後能夠顯著下降。同時,建立博來黴素 (Bleomycin, BLM) 誘導小鼠肺纖維化模式,結果顯示,TMF可以改善小鼠的肺功能,在組織切片蘇木精-伊紅染色 (H&E stain) 可以看到TMF具有保持肺泡完整性的特性,在馬森三色染色 (Masson’ s Trichrome stain) 中, TMF具有減少膠原蛋白沉積的效果,以上結果指出TMF 具有減緩肺纖維化的功效。
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressively deteriorating interstitial lung disease associated with poor clinical prognosis. Currently, treatment options for pulmonary fibrosis are limited, offering little improvement in patient survival. Therefore, the development of novel therapeutic approaches is paramount in IPF research. The pathological features of pulmonary fibrosis include epithelial cell damage, inflammation, myofibroblast formation, and extracellular matrix accumulation. Notably, myofibroblasts play a critical role in fibrosis and are regulated by transforming growth factor-beta (TGF-β). TGF-β presence suppresses myofibroblast apoptosis, leading to abnormal cell proliferation and extracellular matrix production. Thus, attenuating TGF-β-induced fibrogenic effects is crucial for preventing the worsening of pulmonary fibrosis. TGF-β promotes epithelial-mesenchymal transition (EMT), an intricate process in which epithelial cells lose their epithelial characteristics and acquire mesenchymal traits. In pulmonary fibrosis, EMT is characterized by a decrease in epithelial cell markers and an increase in mesenchymal cell markers. Polymethoxyflavones (PMFs) are natural compounds found in citrus peels, known for their high bioactivity and properties such as anti-inflammatory and anti-tumor effects. Among PMFs, 5,7,3',4'-tetramethoxyflavone (TMF) is a major constituent found in Murraya paniculata and Kaempferia parviflora. The aim of this study is investigates the mechanism by which TMF inhibits TGF-β induced idiopathic pulmonary fibrosis. Results demonstrate that stimulation of NIH/3T3 cells with TGF-β (5 ng/mL) increases cell proliferation and the expression of mesenchymal cell markers (α-SMA, Collagen Ⅰ, and Fibronectin), along with the upregulation of EMT-related transcription factor Snail. TGF-β stimulation also significantly enhance the expression of Smad, non-Smad, and Wnt/β-catenin. We found that TMF effectively suppresses Snail mRNA expression, and reduces the expression of Smad, non-Smad, and Wnt/β-catenin. Furthermore, an in vivo bleomycin (BLM)-induced mouse model of IPF shows that TMF improves clinical lung function. H&E staining of tissue sections shows that TMF preserves alveolar integrity. Masson's trichrome staining demonstrates its effectiveness in reducing collagen deposition. These results indicate that TMF possesses the potential to alleviate pulmonary fibrosis.
摘要 i
Abstract ii
目次 iii
表目次 v
圖目次 vi
第一章、 緒論 1
1. 特發性肺部纖維化 (Idiopathic pulmonary fibrosis, IPF) 1
2. 特發性肺部纖維化的病理特徵 1
3. 轉化生長因子β (Transforming growth factor-β, TGF-β) 訊息路徑 2
4. 天然化合物 (Natural Compounds) 3
4.1天然化合物的優勢與應用 3
4.2.多甲氧基類黃酮 (Polymethoxyflavones, PMF) 3
4.3 5,7,3',4'-四甲氧基黃酮 (5,7,3',4'-tetramethoxyflavone, TMF) 4
5. 研究動機 (Motivation) 4
5.1. 體外 (in vitro) 模型 4
5.2. 體內 (in vivo) 模型 4
6. 實驗架構 5
第二章、 材料與方法 6
1. 細胞株種類及培養 (Cell lines and cultures) 6
2. 藥品配製 (Drug preparation) 6
3. 細胞毒殺試驗 (Cytotoxicity assay) 6
4. 西方墨點法 (Western blotting) 7
5. 細胞免疫螢光染色 (Immunofluorescence cytochemistry, ICC) 8
6. 傷口癒合試驗 (Wound healing assay) 8
7. mRNA 表現量的分析 9
7.1. RNA萃取 (RNA extraction) 9
7.2. cDNA 反轉錄 (cDNA reverse transcription) 9
7.3.即時聚合酶連鎖反應(Real-time polymerase chain reaction, RT-PCR) 9
8. 動物實驗 (Animal) 10
9. 博來黴素誘導的小鼠肺纖維化模式 (Bleomycin induced pulmonary fibrosis in mouse model) 10
10. 呼吸道過度反應測試 (Respiratory hyperresponsive assay) 10
11. 組織學染色 (Histology) 11
11.1. 蘇木精-伊紅染色 (Hematoxylin-Eosin Stain, H&E Stain) 11
11.2. 馬森三色染色 (Masson’s Trichrome Stain) 11
12. 小鼠肺臟組織中 mRNA 表現量分析 12
12.1. RNA萃取 (RNA extraction) 12
12.2. cDNA 反轉錄 (cDNA reverse transcription) 12
12.3.即時聚合酶連鎖反應 (Real-time polymerase chain reaction, RT-PCR) 12
13. 體內肝腎功能 (Hepatic and renal functions in vivo) 12
14. 統計分析 (Statistically analysis) 13
第三章、 實驗結果 14
1. TMF 對纖維母細胞毒性、生長與遷移之影響 14
2. TMF 減少TGF-β誘導纖維母細胞活化 14
3. TMF 下調TGF-β誘導纖維母細胞Smad dependent訊息路徑 15
4. TMF 抑制p-Smad3入核與EMT相關轉錄因子 15
5. TMF 抑制TGF-β誘導纖維母細胞 non- Smad dependent訊息路徑 15
6. TMF 減緩博來黴素誘導肺纖維化之小鼠模式 16
7. TMF 對小鼠肝腎功能影響 16
8. TMF 抑制纖維母細胞活化與減輕博來黴素誘導的小鼠肺纖維化的機制模型 16
第四章、 討論 17
參考文獻 21
圖表 27
附錄 54
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