臺灣博碩士論文加值系統

葡萄糖胺(glucosamine)為幾丁聚醣(chitosan)的單體，基本上是一種氨基單醣(amnio monosaccharide)，又稱氨基葡萄醣(2-amino-2deoxy-glucose)，是葡萄醣分子的一個H2O被NH2基所取代。葡萄糖胺(glucosamine)幾乎在所有的生物中都可自然產生，是生物體內會自然產生的眾多monosaccharide的一種。有部分的研究指出glucosamine可以向上調節軟骨細胞(chondrocyte)，減少軟骨基質的降解(degradation)，並同時可以提供軟骨基質，軟骨素(aggrecan)合成的來源。甚至可以向下調控某些inflammatory cytokines，例如：COX-2、IL-1β、TNF-α等發炎相關基因的表現，並且促進成骨細胞的分化。為了探討葡萄糖胺(glucosamine)未來是否可以應用於口腔醫學，本研究的目的是將人類牙周韌帶細胞培養在一般培養液與促進細胞外基質礦化之培養液中，並以添加glucosamine與否設計不同的實驗，以檢測鹼性磷酸酶 (ALP) 活性，成骨指標蛋白的基因表現，及細胞外基質礦化等性質，以探討glucosamine對牙周韌帶細胞的成骨分化之影響。透過體外細胞培養模式，若能確認glucosamine對牙周韌帶細胞的成骨分化具有促進作用，將可以提供後續研究的基礎。
本研究發現：在細胞活性生長測試的部份，以5ug/ml glucosamine培養三天以後即有明顯的促進牙周韌帶細胞的生長。在成骨指標蛋白和基因的表現部份，牙周韌帶細胞經過5ug/mg glucosamine 培養三天之後，Runx2 gene的表現會有明顯上升，但之後隨著培養天數會慢慢向下調控。而ALP 的話，則是在5ug/ml glucosamine 培養七天後ALP的表現有明顯的上升，之後隨著培養天數則會開始慢慢向下調控，到了合併mineralizing medium培養七天後，ALP 的表現反而有下降的趨勢。在細胞基質礦化程度分析的部份，單獨以5ug/ml glucosamine培養七天與十四天皆無法成功誘導牙周韌帶細胞產生基質礦化小體，需要合併mineralizing medium的培養才能進一步誘導牙周韌帶細胞的成骨分化。而合併有5ug/ml glucosamine 以及mineralizing medium一起培養的條件下，glucosamine可以促進牙周韌帶細胞的成骨分化並加快基質礦化小體產生的速度。

Glucosamine is monomer of chitosan, and an kind of amnio-monosaccharide. It is also be called 2-amino-2deoxy-glucose，and is the NH2 group substituted the H2O group of glucose。Some studies reported glucosamine can up-regulate the gene expression in chondrocyte, minimize degradation of cartilage matrix, and supply the source for synthesis of aggrecan. Other studies reported glucosamine can down-regulation some inflammatory cytokines, like COX-2、IL-1β、TNF-α, and also can promote differentiation of osteoblast. In order to investigate if glucosamine can be applied for oral medicine, we cultured human periodontal ligament cells and design different groups of experiments with the supplement of glucosamine or not. We check the ALP activity, the osteogenic-related gene expression, and the extracellular matrix mineralization in glucosamine-treated PDL cell cultures, and investigate the effects of glucosamine on osteogenic phenotype of DL cells.
The results of our study reveal that : Glucosamine can significantly promote the cell proliferation of PDL cells. In the presence of 5ug/ml glucosamine cultured, Runx2 gene of PDL cells is up-regulated, whereas this effect decreased with the culture time. ALP gene expression is up-regulated after 7 days of culture. The presence of 5ug/ml glucosamine in regular medium for 7 and 14 days did not induce the production of mineralized nodules in PDL cells cultures. When mineralizing medium was used, glucosamine can promote the osteogenic differentiation of PDL cells and accelerate the formation of mineralized nodules.

致謝.........................i
中文摘要....................ii
英文摘要....................iv
目錄.........................v
論文索引....................vi
表次目錄....................ix
圖次目錄.....................x

第一章 緒論...........................................1
一、牙周韌帶細胞的異質性與多潛性........................1
二、細胞成骨分化與成骨指標蛋白的基因表現................2
三、葡萄糖胺 (Glucosamine)..............................8

第二章 實驗目的.......................................11

第三章 實驗的材料與方法...............................12
一、人類牙周韌帶細胞的培養..............................12
二、細胞培養液之製備....................................12
三、細胞型態的觀察......................................13
四、細胞生長活性測試 (MTT assay)........................13
五、Total RNA 之萃取及定量..............................14
六、半定量-反轉錄-聚合酶連鎖反應 (RT-PCR)...............15
七、細胞鹼性磷酸酶之染色(Alkaline Phosphatase stain)
與觀察..............................................17
八、細胞鹼性磷酸酶之半定量分析(ALP assay)...............17
九、細胞外基質礦化小體(minerization nodule of
extracellular matrix) 之染色 (Alizain Red stain)
與觀察..............................................18
十、細胞外基質礦化程度的半定量分析(Semi-quatification
of minerization)....................................19
十一、統計方法..........................................20
十二、實驗設計..........................................20

第四章 結果...........................................23
一、Glucosamine對牙周韌帶細胞生長活性的影響.............23
二、Glucosamine對牙周韌帶細胞鹼性磷酸酶表現的影響.......23
三、Glucosamine對牙周韌帶細胞之成骨分化基因表現的影響...23
三.1 以glucosamine培養牙周韌帶細胞3天與7天後之成骨
基因表現.........................................24
三.2 以glucosamine培養7天再進行礦化誘導7天之成骨分
化基因表現.......................................24
四、鹼性磷酸酶染色與活性定量分析........................24
四.1 以glucosamine培養7天後進行鹼性磷酸酶染色與半定
量分析...........................................24
四.2 以glucosamine培養7天後再進行礦化誘導3天及7天的
鹼性磷酸酶的染色與活性定量分析...................25
五、細胞外基質礦外小體的Alizarin red染色(ARS)與半定量分
析..................................................25
五.1 以glucosamine培養7天............................26
五.2 以glucosamine培養14天...........................26
五.3 以glucosamine培養7天後再進行礦化誘導7天.........26
五.4 以glucosamine培養7天後再進行礦化誘導14天........27
第五章 討論...........................................28
一、 Glucosamine對牙周韌帶細胞生長活性的影響............28
二、Glucosamine對牙周韌帶細胞成骨分化指標蛋白及其基因
表現的影響..........................................29
二.1 Runx2 / Cbfa-1..................................29
二.2 ALP.............................................29
三、Glucosamine對牙周韌帶細胞產生基質礦化小體的影響.....31

第六章 結論...........................................34

第七章 未來研究方向...................................35

參考文獻.................................................52

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