牙醫專業學院: 臨床牙醫學研究所指導教授: 鄭景暉;張曉華陳芝宇Chen, Chih-YuChih-YuChen2017-03-062018-07-092017-03-062018-07-092015http://ntur.lib.ntu.edu.tw//handle/246246/277124實驗目的：鹼性纖維母細胞生長因子（Basic fibroblast growth factor, bFGF）在生物體內具有多種功能，並且在細胞生長與分化上扮演重要角色，但其對於牙根尖細胞之影響及訊息傳導路徑還未被完全了解。本實驗之目的在探討bFGF對牙根尖細胞生長與分化的影響，以及其中MEK/ERK訊息傳導路徑的角色。 實驗方法：加入不同濃度bFGF於人類牙根尖細胞做刺激與培養，部分組別加入U0126 （MEK/ERK抑制劑）。透過細胞存活率分析 （MTT assay）、反轉錄聚合酶連鎖反應 （RT-PCR）、西方墨點法 （western blot）、鹼性磷酸酶染色（ALP stain）及免疫螢光染色（immunofluorescent）來檢測bFGF對牙根尖細胞在細胞生長、分化及相關之基因與蛋白質、纖維母細胞生長因子接受器（FGFR）的影響。 實驗結果：bFGF促進牙根尖細胞生長與相關基因及蛋白之表現，包括cyclin B1、cdc2及cdc25c。在成骨/成牙本質分化方面，經過24小時bFGF刺激後，Runx2和osteocalcin表現會增加，但5天之bFGF刺激則會抑制ALP活性。FGFR1、2、3及4均會表現於牙根尖細胞。單獨加入U0126會抑制細胞生長，而合併bFGF使用會降低原本bFGF促進生長之能力。此外，U0126可以抑制因bFGF提升之osteocalcin與TIMP1基因及蛋白表現，而對Runx2與ALP則無影響。U0126亦無法逆轉因bFGF下降之ALP活性。 結論：bFGF對人類牙根尖細胞的影響是很多樣性的，會因藥物刺激的時間而不同，而MEK/ERK在其中亦扮演很重要的角色。本實驗的結果提供我們對bFGF與牙根尖細胞的作用及訊息傳導路徑更進一步的了解，對於未來應用在牙髓壞死之牙齒的根尖生成術與牙髓、牙本質再生也有幫助。Aim : Basic fibroblast growth factor (bFGF) owns multiple biological functions in various tissues, and plays important roles in cell proliferation and differentiation. Human apical papilla cells have been reported to show characteristics of stem cells and are known as stem cells from apical papilla (SCAP). The purpose of this study is to investigate the effects of bFGF on apical papilla cells and the roles of MEK/ERK signaling. We hypothesize that bFGF regulates cell proliferation and differentiation through MEK/ERK. Materials and Methods : Primary-cultured human apical papilla cells were treated under different concentration with or without U0126 (an inhibitor of MEK/ERK). Cell proliferation was measured by MTT assay. The expressions of cell cycle-related and differentiation-related genes and proteins were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. Phosphorylation of signaling molecules was examined by western blot. ALP activity was determined by ALP staining. FGF receptors (FGFRs) were detected by immunofluorescent (IF). Results : In human apical papilla cells, treatment of bFGF (10~500 ng/ml) enhanced the proliferation and the expression of cell cycle-related genes and proteins including cyclin B1, cdc2, and cdc25c. In osteogenic/ dentinogenic differentiation, bFGF promoted the expression of Runx2 and osteocalcin, but ALP activity was suppressed by treatment of bFGF for 5 days. FGFR1, 2, 3 and 4 were expressed abundantly in apical papilla cells. Using U0126 solely decreased the inherent proliferative ability in apical papilla cells, and combined with bFGF inhibited the bFGF-induced enhancement of proliferation and expression of cell cycle-related genes and proteins. The increase of osteocalcin and TIMP1 induced by bFGF was repressed by U0126, while Runx2 and ALP were not changed. Besides, ALP activity attenuated by bFGF could not be reversed by U0126. Conclusion : The effect of bFGF on apical papilla cells is complicated and might be divergent depending on the duration of treatment. MEK/ERK pathway plays important roles in miscellaneous cell functions. These results would be useful for clinical therapies in the future, including apexogenesis and pulp-dentin complex regeneration.2902662 bytesapplication/pdf論文公開時間: 2018/9/24論文使用權限: 同意有償授權(權利金給回饋學校)牙根尖細胞鹼性纖維母細胞生長因子纖維母細胞生長因子接受器MEK/ERK鹼性磷酸?apical papilla cellsbFGFFGFRalkaline phosphatasethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/277124/1/ntu-104-R01422026-1.pdf