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研究生:吳書嫚
研究生(外文):Wu, Shu-man
論文名稱:探討牙釉質衍生物對人類牙齦幹細胞之增生及分化的影響
論文名稱(外文):Effects of enamel matrix derivative on the proliferation and differentiation of human gingival mesenchymal stem cells
指導教授:傅鍔傅鍔引用關係邱賢忠
指導教授(外文):Fu, EarlChiu, Hsien-Chung
口試委員:傅鍔聶鑫涂筱培沈一慶江正陽
口試委員(外文):Fu, EarlNieh, ShinTu, Hsiao-PeiShen, E-ChinChiang, Cheng-Yang
口試日期:2012-05-17
學位類別:碩士
校院名稱:國防醫學院
系所名稱:牙醫科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:74
中文關鍵詞:牙釉基質衍生幹細胞牙齦增生礦化
外文關鍵詞:enamel matrix derivativestem cellgingivaproliferationmineralization
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牙齦幹細胞是分離於牙齦結締組織,學者Winchester於1978年採用低種植密度進行培養細胞,成功培養出幹細胞。牙齦幹細胞經實驗證實有跨胚層分化的潛力,分離牙齦幹細胞後,將細胞培養於各種誘導粉為的培養基中,包括:成骨、神經和脂肪細胞的分化培養基。然後再使用牙釉質衍生物(EMD)處理,來探討它對細胞增殖和分化的影響。MTS實驗是使用牙釉質衍生物的處理24小時和48小時,去分析細胞增生的情形,牙釉質衍生物濃度分別為0、25、100 μg/mL。之後評估細胞的礦化情形,使用茜素紅S(ARS)的染色,時間為7、14、21、28天培養,使用成骨培養基培養。將用逆轉錄聚合酶鏈式反應(RT-PCR)評估有關骨分化的標記,如core binding factor alpha (cbfa-1)、Alkaline phosphatase (ALP)和 bone γ-carboxyglutamate (Gla) protein (BGLAP, osteocalcin)的一些骨鈣化基因,細胞培養時間為1、2、3、4週。此外,也利用牙齦幹細胞與Bio-Oss骨粉混和之移植物種入動物背部皮下以觀察其對於骨組織生成之影響。分別從這些分析結果,我們觀察到牙釉質衍生物對人類牙齦分離出的幹細胞分化之影響。
The gingival stem cells will be isolated from the gingival specimens of the patients received crown lengthening procedures. The gingival stem cells will be obtained by the low density culture system method described by Winchester (1978). To investigate the potential of undergoing multiple-lineage differentiation for the gingival stem cells, the isolated gingival stem cells will be supplemented with various induction media, including the osteogenic differentiation medium, and the neural and adipogenic differentiations.
Then, the effects of enamel matrix derivative (EMD) on the cell proliferation and differentiation of the cells will be examined. The MTS assay will be selected to examine the cell proliferation after the EMD stimulation (with the concentrations of 0, 25, 100 μg/mL) for 24 and 48 hours. To evaluate the mineral induction of the cells, the tests of the Alizarin red S (ARS) staining and Calcium releasing assay will be performed after 7, 14, 21, 28 days of cell culturing in an osteogenic medium. Some of the critical genes related to mineral differentiation of cells, such as core binding factor alpha (cbfa-1), Alkaline phosphatase (ALP), and bone γ-carboxyglutamate (Gla) protein (BGLAP, osteocalcin), will be evaluated by reverse transcription polymerase chain reaction (RT-PCR) after 1, 2, 3,and 4 weeks of cell culturing, respectively.
Furthermore, the transplants which were combined with GMSC and Bio-Oss was transplanted in dorsal subcutaneous space of rats to examine the effects of osteogenesis. The results from this study will let us understand the effects of EMD on the osteogenic differentiation of the stem-like cells isolated from the human gingiva.

第一章、緒論及目的 2
第一節、幹細胞於牙科之發展 2
第二節、牙釉基質衍生物 10
第三節、研究目的 12
第二章、材料與方法 15
第一節、實驗驗證材料 15
第二節、細胞實驗驗證 19
第三節、動物實驗驗證 26
第四節、統計學分析 31
第三章、實驗結果 33
第一部份、細胞實驗驗證 33
第二部份、動物實驗驗證 36
第四章、實驗討論 39
第一節、EMD對細胞增生及分化之影響 39
第二節、骨標記在骨生成的意義 40
第五章、結論 42
第六章、參考文獻 62


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