- 學位論文
FAK在眼角膜上皮細胞傷口癒合之角色
The role of focal adhesion kinase in corneal epithelial wound healing
摘要
Integrins 是一種於細胞膜上的受體，可與細胞外的基質 (extracellular matrix) 進行交互作用，調控細胞的黏著、移動和生長等細胞功能。 Focal adhesion kinase (FAK)是一種分子量125 kDa的酪胺酸激脢 (tyrosine kinase) ，經由與 talin 和 paxillin 等 focal adhesion complex 的蛋白質間交互作用，在 integrin 於細胞內部的一端形成聚合調控細胞遷移與增生的能力，扮演 integrin 的細胞訊息傳遞中重要的訊息分子。本研究中主要以基因剔除和基因轉殖小鼠做實驗，包含眼角膜專一性剔除 FAK 的小鼠 (K12-rtTA/tetO-Cre/ FAK f/Δ) 和 Akt 基因剔除小鼠來探討 FAK 與 Akt 在眼角膜上皮細胞增生與傷口癒合所扮演的角色。實驗結果可知， FAK 基因剔除會導致減慢眼角膜上皮細胞早期傷口癒合修復的速度，而且不同於 Akt 基因剔除小鼠會影響了晚期的傷口癒合。不僅如此，如果以 infection 的方式大量表現 FAK 這蛋白質於 FAK 基因剔除的小老鼠眼角膜上，可因此回復傷口癒合的速度。而實驗中藉由使用人類的角膜上皮細胞 (HCECs) 做 in vitro 傷口癒合的測試，發現當眼角膜上皮細胞受到細胞外機質 (collagen typeIV) 和 EGF 的刺激下， FAK 蛋白的表現以及活化有可能參與在調控細胞遷移的部份，這部份的實驗便印證了 in vivo 實驗的結果。除此之外，藉由加入不同 MAPK pathway 的抑制劑，例如 : PP2 (Src抑制劑)、SP600125 (Ｅrk抑制劑) 和 SB203580 (p38抑制劑)，我們發現 FAK 會藉由調控下游的 Src 和 p38 信息傳遞路徑來調控眼角膜上皮細胞的傷口癒合。所以總合上的研究就可以比較清楚了解 FAK 在眼角膜上皮細胞傷口癒合時所扮演的生理機能與角色。
並列摘要
Given the importance in several cellular processes, including cell migration and proliferation, focal adhesion kinase (FAK) has attracted much interest on its role in physical and pathological relevance. The biological role of FAK is emerging to be elucidated due to the availability of genetic engineered mice, in particular the FAK floxed mice, by combining with and varied Cre mice, it allows us to examine the functionality of FAK in physiopathologic conditions which are precluded due to the embryonic lethality of the conventional FAK knockout. Taking advantage of the Cre/loxP driven FAK conditional knockout approach, we generated the K12-rtTA/tetO-Cre/ FAK f/Δ triple transgenic mice to specifically delete FAK in corneal epithelial cells (CE-FAK), which consequently resulted in an impairment of corneal epithelial wound healing compared with the control mice. The impaired wound healing in corneal epithelial cells predominantly occurred in the earlier stage of the healing process in contrast to the prominent delay of wound healing in the late stage in AKT1-/- mice. Furthermore, the defect in the corneal epithelial wound healing of CE-FAK mice was reversed by infection/overexpression with adenovirus-bearing wild type FAK but not Y397F or FRNK mutant. In agreement with the above in vivo result, we also demonstrated several analogous in vitro results using human corneal epithelial cells (HCECs), highlighting that increased FAK expression and activation are concurrently associated with the promotion of cell adhesion and migration toward collagen IV and EGF. These results indicate a pivotal role for FAK and its mediated signaling in response to corneal epithelial wound stimuli to promote its healing. In addition, via applying several pharmacological inhibitors, such as PP2 (an Src inhibitor), SP600125, and SB203580, we also found that Src and p38 are potential downstream effectors of FAK-mediated wound healing in corneal epithelial cells in mice. Taken together, this study provides comprehensive data for an important role of FAK in corneal epithelial wound healing and underscores the need for FAK in physiopathological maintenance.
參考文獻
延伸閱讀
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