轉型生長因子β (Transforming Growth Factor-β, TGF-β)是一種多功能細胞激素，影響廣泛生理功能，在細胞當中TGF-β及其訊息傳遞須受嚴格控制，TGF-β訊息傳遞起始於Type II TGF-β receptor (TβRII) 和TβRI 結合後活化下游Smad蛋白質，將訊息傳入細胞核後控制基因表現。在癌症發展過程中TGF-β有著截然不同的調控，於初期癌症中TGF-β會抑制細胞生長已達到抑制腫瘤發展的過程，在癌症發展晚期時，TGF-β會促進細胞增生並加強癌細胞的入侵能力，除此之外，TGF-β也被證實和其他疾病息息相關，例如:器官纖維化和自體免疫疾病，因此TGF-β訊息傳遞的小分子抑制劑可以作為上述疾病有淺力的藥物。Pentabromopseudilin (PBrP)是一種天然的氯吡咯衍生物，目前可以人工合成，在哺乳類動物細胞當中可以作為Myosin V蛋白質的抑制劑。在本篇研究當中，我們發現PBrP 可以加速TβRII 被降解，以此降低細胞對TGF-β的反應，PBrP 會抑制TGF-β誘導的SMAD蛋白質磷酸化與PAI-1報導基因啟動子活化，半抑制濃度(half maximal inhibitory concentration, IC50)介於0.05到0.1 μM之間，在A549和HepG2細胞當中PBrP 會抑制EMT (epithelial-to-mesenchymal transition)的發生過程，並減弱細胞的爬行能力。經由實驗證實PBrP 會使細胞膜表面TβRII由lipid raft區域經胞吞作用進入細胞後送至溶媒體降解，使細胞膜表面可接收TGF-β訊息的受氣數量降低，最後造成細胞對TGF-β的反應下降。

Transforming Growth Factor-β (TGF-β) is a multifunctional cytokine that involved in many biological processes. TGF-β signaling transduction initiates when binding to Type II TGF-β receptor (TβRII). Once binding to TGF-β, TβRII forms heterocomplex with Type I TGF-β receptor (TβRI) and phosphorylates Smad2 and Smad3. P-Smad2/3 then forms a homotrimer with Smad4. Finally, R-Smad/Smad4 complexes are then translocated into the nucleus where they act as a transcriptional regulator of target genes. TGF-β signaling plays an opposite role during carcinoma progression. In the early stage, TGF-β acts as a tumor suppressor through inhibiting cell proliferation. Conversely, TGF-β promotes tumor cell proliferation and enhances mobility of cell in the late stage of carcinoma. In addition, TGF-β also is associated to other diseases such as organ fibrosis and autoimmune diseases. Pentabromopseudilin (PBrP) is a natural chlorinated phenylpyrrole compound that shows a broad range of antimicrobial activity. In mammalian cells, PBrP acts as an allosteric inhibitor of myosin 1c (Myo1c). In this study, we find that PBrP attenuates TGF-β responsiveness by promoting degradation of surface TβRII. Furthermore, PBrP inhibits TGF-β-induced Smad2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) promoter activation with a IC50 betewwn 0.05 μM to 0.1μM. Abolishing the process of epithelial-to-mesenchymal transition (EMT) in A549 and HepG2 cells, PBrP is found to suppress the mobility of cells. The results also demonstrate that PBrP directs surface TβR-II translocation to non-raft fractions from lipid raft. The translocation is followed by internalization and degradation of TβRII via lysosomal pathway. By accelerating TβR-II degradation, PBrP inhibits TGF-β-stimulated cellular responsiveness.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
縮寫表 vi
前言 1
材料 8
一、細胞株 8
二、細胞培養(Cell culture) 8
三、質體備製 (Plasmids) 8
四、真核細胞轉染 (Transfection) 8
五、西方墨點法 (Western Blotting) 8
六、免疫螢光染色法 (Immunofluorescence) 9
七、蔗糖濃度梯度離心 (Sucrose Gradient Centrifugation) 9
八、螢火蟲冷光活性分析 (Luciferase Activity Assay) 10
九、細胞核萃取 (Nuclear Extract) 10
十、細胞膜表面受器標定及胞吞試驗 (Biotinylation and Endocytosis Assays) 10
十一、細胞基因剔除 10
實驗方法 11
一、細胞培養 (Cell Culture) 11
二、質體製備 11
三、真核細胞轉染 (Transfection) 11
四、西方墨點法 (Western Blot) 12
五、免疫螢光染色法 (Immunofluorescence staining) 13
六、蔗糖濃度梯度離心 (Sucrose Gradient Centrifugation) 13
七、螢火蟲冷光活性分析 (Luciferase Activity Assay) 14
八、細胞核萃取 ( Nuclear Extract ) 14
九、細胞膜表面受器標定及胞吞試驗 (Biotinylation and Endocytosis Assays) 14
十、細胞基因剔除 14
十一、傷口癒合實驗 15
結果 16
PBrP減弱TGF-β/Smad訊息傳遞 16
PBrP會抑制Smad2/3的核轉移 16
PBrP會抑制TGF-β誘導的轉錄活性 17
PBrP會抑制TGF-β誘導的EMT和細胞間基質製造 18
PBrP會干擾TGF-β誘導的細胞爬行 19
PBrP透過促進TβRII降解以抑制TGF-β訊息傳遞 19
剔除Myosin-Va基因會降低TβRII數量與TGF-β訊息傳遞 21
PBrP透過溶酶體使TβRII降解 21
PBrP促進TβRII經由lipid-raft區域降解 23
討論 44
參考文獻 48
補充圖表 53

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