臺灣博碩士論文加值系統

　　過去的研究已知肝細胞生長因子(HGF)可誘導培養於第一型膠原蛋白(Collagen)凝膠中的狗的腎臟上皮細胞株-Madin-Darby canine kidney (MDCK) 細胞形成分支管狀的結構。而細胞的分裂增生，移行，細胞間的交互作用，細胞生存及分化等則是構成管狀結構形成的基本要素。然而，肝細胞生長因子刺激MDCK細胞形成管狀結構包含哪些細胞訊息路徑和蛋白質則尚未被研究透徹。轉錄因子中的Stat3已被證實可被肝細胞生長因子所活化並參與部分腎管形成的過程，然而其在分支管狀結構形成過程中扮演何種重要角色則仍不清楚，因此我們利用在MDCK細胞中大量表現持續活化型Stat3 (Stat3-C) 及顯性抑制型Stat3 (Stat3Y705F)的細胞株來釐清這個問題。藉由測試與比較不同細胞株間的生理功能，我們發現Stat3-C抑制而Stat3Y705F卻可促進分支管狀結構的形成。Stat3-C亦可調節第一型膠原蛋白所誘導的細胞移行能力及細胞與細胞間的結合強度，而Stat3Y705F則會促進纖維蛋白(fibronectin)所誘導的細胞移行能力。雖然Stat3並不影響到細胞在正常培養狀態下的分裂生長，然而Stat3Y705F大量表現的細胞株卻在惡劣的培養條件下具有較好的存活率，包括低濃度血清合併低密度細胞培養、第一型膠原蛋白內培養與不著地生長的環境。由此可知，Stat3Y705F可藉由增強細胞與細胞間的結合強度及存活率而達到促進肝細胞生長因子誘導分支管狀結構的形成。更進一步證實第一型膠原蛋白所誘導的Stat3 tyrosine 705磷酸化可被Stat3Y705F競爭性的抑制，暗示Stat3調節的轉錄作用與第一型膠原蛋白所誘導的細胞移行能力有關。另一方面，大量表現Stat3Y705F的細胞亦同時具有高量的Stat3 serine 727的磷酸化、纖維蛋白所誘導的細胞移行能力及在1% FCS培養條件下有較好的細胞存活率。由此可知，Stat3 serine 727的磷酸化和細胞的存活率有關。此外，我們也發現Stat3可負向調節Bcl-xL的蛋白質表現量及正向調節Pyk2的蛋白質表現量。是否這些蛋白質與Stat3所調節的細胞存活率與細胞移行有關則需要進一步的探討。

　It has been well established that hepatocyte growth factor (HGF) induced the branching tubule formation of Madin-Darby canine kidney (MDCK) cells cultured in type I collagen gel. Tubulogenesis per se requires the involvement of cell proliferation, migration, cell-cell association, anti-apoptosis and differentiation. Although the transcription factor of signal transducers and activators of transcription 3 (Stat3) is involved in HGF-induced signaling pathways required for epithelial tubule formation, the specific role of Stat3 in tubulogenesis is still unclear. To clarify the role of Stat3 in HGF-induced tubulogenesis, Stat3-C (constitutive active Stat3) and Stat3Y705F (dominant-negative (DN) Stat3) were transfected into MDCK cells. The assessment of biologic functions, including cell proliferation, migration, cell-cell dissociation, tubule formation, and anchorage-independent growth was made. Our results showed that active Stat3 inhibited, but DN-Stat3 facilitated HGF-induced tubule formation in collagen gel. Active Stat3 regulated collagen-induced cell migration and cell-cell dissociation, whereas DN- Stat3 promoted fibronectin-induced cell migration. Although Stat3 did not affect cell proliferation when cells were cultured under normal conditions, DN-Stat3 prevented cell death induced by 1% fetal calf serum (FCS), type I collagen gel, and agarose gel. Therefore, DN-Stat3 promoted HGF-induced tubulogenesis via enhancement of cell association as well as survival. Furthermore, collagen induced tyrosine phosphorylation of Stat3 that was down regulated by DN-Stat3, indicating that Stat3-mediated transcription may be associated with collagen induced migration capability. In addition, cells harboring DN-Stat3 exhibited higher levels of Stat3 serine phosphorylation, fibronectin-induced migration and survival under 1% FCS, suggesting that serine phosphorylation of Stat3 is associated with cell survival. Moreover, we also found that Stat3 down-regulated expression of Bcl-xL and up-regulated expression of Pyk2. Whether these proteins are involved in Stat3-regulated survival and migration remains to be determined.

Content
Abstract I
中文摘要 III
誌謝 V
Content VI
Figure content VII
Introduction 1
Materials and Methods 7
Results 17
Discussion 27
References 34
Figures
Table 1
Appendix A
Appendix B
作者簡歷

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