- 學位論文
褪黑素對於牙根尖細胞生物功能之影響
Effect of Melatonin on the Biological Activity of Human Apical Papilla Cells
摘要
實驗目的：褪黑素因具有多種不同的生物學功能，已經被廣泛應用於幹細胞和組織工程等研究領域。而根據先前的研究，人類牙根尖細胞 (apical papilla cells) 顯示出具有幹細胞的特徵跟能力，並被稱為根尖乳突幹細胞 (Stem cells from the apical papilla, SCAP)。本研究的目的是想探討褪黑素對牙根尖細胞的影響以及PKA和PKC信號通路的作用。實驗方法：本實驗用不同濃度的褪黑素來刺激與培養牙根尖細胞，部分組別加入H89 (PKA抑制劑) 或H7 (PKC抑制劑) 做預處理。再以顯微鏡觀察細胞形態，並用MTT 做細胞存活率分析。使用反轉錄聚合酶連鎖反應 (RT-PCR)、西方墨點法(western blot) 檢測褪黑素的受體表現，還有齒原細胞和成骨細胞分化相關之基因跟蛋白的表現。也透過西方墨點法和免疫螢光染色 (immunofluorescent) 染色檢測信號分子的磷酸化。並使用 phalloidin 染色檢測肌動蛋白絲。實驗結果: 在人類牙根尖細胞中，有表現褪黑素受體MT1、MT2、MT3和RORα。加入褪黑素處理會增強成骨相關的轉錄因子Runx2和SP7的表現。對於跟細胞移動有關的作用，我們發現褪黑素會促進cofilin的磷酸化並刺激肌動蛋白絲聚合。但褪黑素並沒有改變cofilin-1本身的基因和蛋白表現。而H7會抑制褪黑素誘導的cofilin磷酸化，H89則反而更刺激磷酸化cofilin的表現。結論：這是首次檢測出人類牙根尖細胞中有表現褪黑素受體的研究。因為褪黑素對牙根尖細胞的影響是複雜的，並且可能會根據給藥物的劑量和作用時間而不同。值得進一步深入研究褪黑素的相關生物功能，這將會對未來的臨床治療很有幫助，無論是在再生牙髓病學領域或是骨頭再生跟重建的領域。
並列摘要
Aim: Melatonin owns multiple biological functions in various tissues, and becomes widely used in multiple areas including stem cell biology and tissue engineering. 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 melatonin on apical papilla cells and the role of PKA and PKC signaling pathways. Materials and methods: Primary-cultured human apical papilla cells were treated with different concentrations of melatonin and also with/without H89 (an inhibitor of PKA) or H7 (an inhibitor of PKC) pretreatment. Microscope observed cell morphology. Cell proliferation was measured by MTT assay. The expression of melatonin receptors, odontogenic/osteogenic transcription factors, differentiation markers, and actin depolymerizing factors were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot. Phosphorylation of signaling molecules was examined by western blot and immunofluorescent (IF) staining. Actin filaments were detected by phalloidin staining. Results: In human apical papilla cells, the expression of melatonin receptors, MT1, MT2, MT3/NQO2, and RORα were found. Treatment with melatonin (10~500 μg/ml) enhanced the expression of osteogenic transcription factors, Runx2 and SP7. In cell migration and morphogenesis, melatonin increased the phosphorylation of cofilin and stimulated actin filament polymerization. However, the mRNA and protein levels of cofilin1 were not changed by melatonin treatment. The enhancement of cofilin phosphorylation which induced by melatonin was repressed by H7, while H89 even stimulated the expression of p-cofilin. Conclusion: This is the first study to detect the expression of melatonin receptors in human apical papilla cells. However, the effect of melatonin on apical papilla cells is complicated and might be divergent depending on the dose and duration of treatment. Further investigating the biological activity of melatonin would be useful for clinical therapies in the future, including regenerative endodontics and bone regeneration.