轉型生長因子(Transforming growth factor β1, TGF-β1) 是多胜肽類生長因子家族中的一員，並且參與許多導致腫瘤病變的生物學過程。包括細胞增殖、細胞外基質分泌、細胞貼附、移動、分化和凋亡等。而TGF-β1的訊息傳遞會經由細胞膜上的兩個蛋白質接受器Type I TGF-β receptor (TβR-I) 和Type II TGF-β receptor (TβR-II) 。TGF-β磷酸化Samd 2/3，被磷酸化的Samd2/3會轉移到細胞核內和其他轉錄因子一起調控標的基因的轉錄。TGF-β在癌症的早期階段會藉著抑制細胞增生以及促進細胞凋亡的方式抑制著腫瘤發展。然而在晚期階段TGF-β則會促進腫瘤細胞的生長、侵襲、轉移以及幫助腫瘤細胞逃過免疫系統攻擊。一些能夠抑制細胞TGF-β訊息傳遞的小分子抑制劑，對於治療癌症的研究上有很大的幫助。在研究訊息傳遞路徑間的交互作用上，小分子抑制則是一種很有力的工具。在我們的實驗中，發現一種從美麗海綿屬Pseudoceratina sp.萃取出二溴酪氨酸的衍生物(1′R,5′S,6′S)-2-(3′,5′-dibromo-1′,6′-dihydroxy-4′-oxocyclohex-2′-enyl) acetonitrile (DT)，經由抑制TβR-I上的絲氨酸/蘇氨酸激酶，來抑制TGF-β下游訊息的傳遞。為了瞭解DT對TGF-β的訊息傳遞的影響，我們使用了冷光酵素活性分析、西方墨點法、傷口癒合試驗和試管內Activin receptor-like kinase (ALK5)激酶活性試驗等分析方法來研究DT對TGF-β的訊息傳遞的影響，並且使用上皮細胞來研究DT對TGF-β引發上皮細胞間質轉化的抑制作用。在研究中發現，這新型的ALK5抑制劑能有效的抑制TGF-β刺激的Smad2/3磷酸化和Smad2/3的核轉移。另外DT還能抑制TGF-β刺激的上皮細胞間質轉化現象和A549細胞的遷移。我們的研究結果表示DT在未來可以應用在纖維化疾病和癌症的治療。

The Transforming growth factor β1 (TGF-β1) is belong to transforming growth factor superfamily. Many tumor lesions process are related to TGF-β1, such as: cell proliferation, extracellular matrix secretion, cell attachment, movement, differentiation and apoptosis. TGF-β1 cell signaling via two protein receptors on membrane which are Type I TGF-β receptor (TβR-I) and Type II TGF-β receptor (TβR-II). TGF-β is activated that lead smad2/3 to phosphorylation, and p-Smad2/3 will transfer to nuclear than regulates the transcription of the target gene with other transcription factor. At cancer early stage, TGF-β will use inhibit cell proliferation and promote cell apoptosis to inhibit cancer growth, but at cancer late stage,instead, TGF-β will promote cancer cell growth, invasion, transfer and help it to escape the immune system attack. There are some small molecule inhibitors which can inhibit TGF-β cell signal transduction have great value at cancer research. Small molecule inhibitor is a powerful tool in research of signal transduction pathway interaction. In this study, we found (1′R,5′S,6′S)-2-(3′,5′- dibromo-1′,6′-dihydroxy-4′-oxocyclohex-2′-enyl) acetonitrile (DT), a bromotyrosine derivate from Pseudoceratina sp., which inhibits the TβR-I serine/threonine kinase then inhibits TGF-β downstream cell signaling. We use such as: luciferase activity assay, western blotting, wound healing assay, in vitro ALK5 kinase assay etc. to know the effect of DT on TGF-β cell signaling, and use epithelial cells to study of the inhibitory effects of DT on TGF-β-induced Smad signaling and epithelial-to-mesenchymal transition. We also confirmed the new ALK5 inhibitor can effectively inhibit TGF-β stimulate smad2/3 to phosphorylation and inhibit smad2/3 transfer to nuclear. In addition, DT also can inhibit TGF-β stimulate epithelial-to-mesenchymal transition and A549 cell metastasis. Our study showed DT can apply to treatment of fibrotic diseases and cancer in the future.

目 錄
論文審定書…………………………………………………………………………… i
致謝…………………………………………………………………………………... ii
摘要………………………………………………………………………………….. iii
Abstract……………...……………………………………………………………… iv
目錄……………………………………………………………………...…..….…… vi
圖次………………………………………………………………………........…… viii
縮寫表………………………………………………………………...…………...… xi
前言
　 TGF-β.................................................................................................................... 1
TGF-β和癌症之間的關係.................................................................................... 3
　 TGF-β信號傳遞的小分子抑制劑對癌症治療的影響........................................ 5
DT.......................................................................................................................... 6
材料與方法
一、藥品與化學試劑............................................................................................. 8
二、質體與抗體................................................................................................... 10
三、細胞培養....................................................................................................... 11
四、細胞毒性試驗 (MTT Assay)……………………………………………... 12
五、電穿孔法(Electroporation)............................................................................ 12
六、冷光酵素活性分析(Luciferase Activity Assay)........................................... 13
七、西方墨點法(Western Blotting).................................................................... 13
八、核蛋白萃取(Nuclear Protein Extract).......................................................... 14
九、RNA抽取(RNA Isolation)和反轉錄PCR (Reverse Transcription-PCR, RT-PCR)....................................................................................................... 15
十、聚合酶連鎖反應(Polymerase Chain Reaction, RCP)和洋菜膠電泳法(Agarose gel Electrophoresis)..................................................................................... 16
十一、免疫螢光染色(Immunofluorescence)..................................................... 17
十二、傷口癒合試驗(Wound Healing Assay)................................................... 17
十三、試管內ALK5激酶試驗(In Vitro ALK5 Kinase Assay)......................... 18
十四、統計分析……………………………………………………………….. 18
結果
DT抑制TGF-β刺激Smad2/3的磷酸化.......................................................... 19
DT抑制p-Smad2/3 轉移至細胞核.................................................................. 20
DT抑制TGF-β所誘導的轉錄作用.................................................................. 21
DT通過抑制ALK5的活性來調控TGF-β訊息傳遞路徑.............................. 22
DT減弱TGF-β誘導上皮細胞間質轉化.......................................................... 22
DT抑制TGF-β所誘導的細胞遷移.................................................................. 23
討論............................................................................................................................. 24
參考文獻..................................................................................................................... 62

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