| 摘要: | 氣道纖維化在嚴重氣喘中扮演著關鍵的角色，纖維化是一個漸進且無法逆轉過程。過去的研究發現，yes-associated protein (YAP)過度表現會使collagen、fibronectin及connective tissue growth factor (CTGF)的表現增強，促使肺纖維母細胞增殖、遷移及分化為肌纖維母細胞使extracellular matrix生成沉澱，引發肺纖維化。已知a disintegrin and metalloprotease 17 活化能將細胞膜外的 preadipocyte factor 1 (Pref-1)切除，釋放出具活性50 kDa的Pref-1，Pref-1經由Notch或Wnt路徑來調控脂肪新生及肝纖維化。Src可透過YAP調控纖維母細胞活化。在先前我們研究發現，Pref-1可增加人類肺部纖維母細胞CTGF表現增加。然而，Pref-1是否藉由α5β1 integrin receptor/Src路徑活化YAP調控肺纖維母細胞的CTGF表現仍不清楚。我們研究結果顯示，嚴重氣喘病人的氣道組織當中有大量YAPY357蛋白表現，且Ovalbumin誘導氣喘的小鼠模型中，dlk1纖維母細胞條件剃除小鼠可以減少OVA誘導YAPY357磷酸化和Src表現。在人類肺纖維母細胞中Pref-1刺激YAPY357及YAPS127磷酸化增加，我們也發現使用YAP siRNA可以抑制Pref-1誘導纖維母細胞CTGF表現，Pref-1誘導YAP進入細胞核並結合至CTGF的啟動子。此外，Pref-1能誘導肺纖維母細胞SrcY416磷酸化，PP1 (Src抑制劑)可減少Pref-1刺激YAPY357及CTGF的表現。透過Integrin alpha 5 siRNA抑制實驗發現可以抑制Pref-1活化YAPY357及抑制CTGF表現。綜上所述，我們推測Pref-1可經由α5β1 integrin/Src/YAP路徑調控人類肺纖維母細胞CTGF的表現。因此，本碩士論文探討Pref-1透過α5β1 integrin receptor/Src/YAP傳遞路徑活化肺纖維母細胞CTGF的表現，希望透過了解Pref-1在氣道纖維化中的機轉，在未來對於治療嚴重氣喘及氣道纖維化的發展能夠有所幫助。
關鍵詞：嚴重氣喘；氣道纖維化；人類肺纖維母細胞；Pref-1；YAP；CTGF
Airway fibrosis plays a crucial role in severe asthma, which is a progressive, irreversible process. Previous studies have indicated that overexpression of yes-associated protein (YAP) enhances the expression of collagen, fibronectin, and connective tissue growth factor (CTGF), promoting proliferation, migration, and differentiation of lung fibroblasts into myofibroblasts, leading to extracellular matrix deposition and subsequent pulmonary fibrosis. Activation of a disintegrin and metalloprotease 17 is known to cleave Preadipocyte factor 1 (Pref-1) from the cell membrane, releasing its active 50 kDa form. Pref-1 regulates adipogenesis and hepatic fibrosis via the Notch or Wnt pathways. Src activates fibroblasts through YAP regulation. In our previous studies, Pref-1 increased CTGF expression in human lung fibroblasts. However, it remains unclear whether Pref-1 activates YAP to regulate CTGF expression in human lung fibroblasts via the α5β1 integrin receptor/Src pathway. Our research founds abundant expression of phosphorylated YAPY357 protein in airway tissues of severe asthma patients. In an ovalbumin-induced asthma mouse model, fibroblast specific deletion of dlk1 reduced OVA-induced YAPY357 phosphorylation and Src expression in airway tissue. Stimulation with Pref-1 in human lung fibroblasts increased phosphorylation of YAPY357 and YAPS127. Additionally, YAP siRNA inhibited Pref-1-induced CTGF expression, and Pref-1 induces YAP nuclear translocation and binding to the CTGF promoter. Furthermore, Pref-1 induced SrcY416 phosphorylation in lung fibroblasts. PP1 (a Src inhibitor), and Integrin α 5 (ITGA5) siRNA inhibited Pref-1-induced YAPY357 activation and CTGF expression. In summary, we propose that Pref-1 regulate CTGF expression in human lung fibroblasts via the α5β1 integrin/Src/YAP pathway. Therefore, this thesis explores how Pref-1 activates CTGF expression through the α5β1 integrin receptor/Src/YAP signaling pathway in human lung fibroblast, aiming to contribute to the understanding of Pref-1 mechanisms in airway fibrosis, potentially aiding future developments in severe asthma and airway fibrosis therapies.
Key words：severe asthma, airway fibrosis, human lung fibroblasts, Pref-1, YAP, CTGF |
