臺灣博碩士論文加值系統

牙齒的發育主要透過口腔上皮及來自神經脊的間葉細胞，兩者之間不斷發生訊息傳遞，口腔上皮最後分化為牙釉質母細胞，形成牙釉質；來自神經脊的間葉細胞最後分化為牙齒其他的組織。一旦牙齒萌發，即喪失口腔上皮的來源，這是人類無法再生牙齒的原因。
2006年，Yamanaka 利用基因轉染技術將體細胞重新編譯 (reprogramming)，成為誘導性多功能幹細胞 (iPSCs)，其細胞表現出胚胎幹細胞特有的標記基因與類似的生長形態，可分化成三種胚層的細胞，並且保有原來細胞的特性。所以本實驗利用這項技術，將來自牙髓的細胞重新編譯成 iPSCs，再與牙齒成體間質幹細胞共培養，希望能重新開啟胚胎發育早期口腔上皮與其下間質組織間的相互訊息傳遞。
另一方面，iPSCs 建立了個人化的細胞來源，可利用 iPSCs 研究一些缺乏實驗動物平台的人類疾病，進而做藥物篩檢與開發，建立個人化的醫療平台。本實驗以貝斯特氏症 (Best disease) 為例，取得此病患的牙髓細胞，將其重新編譯為 iPSCs，並與正常人比較，其中兩種蛋白質：Bestrophin-1 和 STXBP2 在患病的細胞中表現量明顯低於正常未患病者，接下來亦將進一步探討此現象與疾病之間的關聯性。

The organogenesis of tooth starts with the thickening of oral epithelium. Subsequent to the increase in thickness, oral epithelial cells initiate the reciprocal signaling with the underlying ectomesenchyme. The epithelial cells differentiate into ameloblasts to form enamel while the ectomesenchymal cells differentiate into other cells to form the remaining tooth structures. However, the regeneration of enamel is still not feasible because the enamel epithelium is degenerated once the enamel formation is completed and leaves no ameloblasts in situ for further manipulation. Also the whole tooth regeneration is not possible without the embryonic epithelium to initiate the reciprocal signaling activities.
In 2006, the discovery of genomic reprogramming of human somatic cells to embryonic stem cells (ESCs)-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem cells (iPSCs), possess many similar properties as ESCs while keep the original cells’ characteristics. Therefore, it is expected that dental cell-derived iPSCs own higher potential than others in regenerating tooth structures. In this study, we are going to create iPSCs from human dental pulp cells first. Created iPSCs will be induced into an ectoderm cell lineage and then cocultured with dental stem cells of mesenchymal origin. The goal is to recreate the reciprocal signaling events happening between original dental epithelium and mesenchyme to successfully regenerate a complete tooth.
On the other hand, iPSCs create the personal cell origin. We can use iPSCs to investigate human diseases which lack of experimental animal platforms. Thus, we can study of drug screening and development, and create the personalized medical platform. In this study, we were going to investigate Best disease. We obtained the patient’s dental pulp cells, reprogrammed into iPSCs (Best-iPSCs), and compared with the iPSCs from normal person. In Best-iPSCS, these two proteins: Bestrophin-1 and STXBP2, had expression levels significantly lower than non-diseased iPSCs. In the future, we are going to further investigate this phenomenon and their correlation with the Best disease.

目　　錄
致謝 II
中文摘要 III
英文摘要 IV
目錄 VI
圖表目錄 VIII
壹、緒論 1
　一、牙齒的發育 1
　二、幹細胞 3
　三、誘導型多功能性幹細胞 5
貳、材料與方法 7
　一、牙髓細胞的樣本來源及培養 7
　二、培養人類胚胎幹細胞 7
　三、產生誘導型多潛力幹細胞 8
　　3-1反轉錄病毒的生產 8
　　3-2反轉錄病毒感染 9
　四、三胚層分化 9
　五、免疫螢光染色 10
　六、即時定量聚合酶連鎖反應 11
　　6-1 RNA 的萃取 11
　　6-2反轉錄聚合酶鍊鎖反應 12
　　6-3即時定量聚合酶連鎖反應 12
　七、流式細胞儀 13
参、結果 14
肆、討論 34
伍、參考文獻 36

 
圖　表　目　錄
圖一、分離牙髓細胞 17
圖二、牙髓細胞的型態 18
圖三、利用流式細胞儀檢測牙髓細胞的細胞表面特定標記 19
圖四、利用免疫螢光染色比較牙髓細胞、骨髓細胞和上皮細胞蛋白質的表現情形 20
圖五、檢測牙髓細胞其內生性 pluripotent 基因的表現量 21
圖六、將牙髓細胞重新編譯 (reprogramming) 的過程 22
圖七、比較胚胎幹細胞與來自牙髓的 iPSCs，其型態與幹細胞特性的標記蛋白 23
圖八、利用免疫螢光染色檢測 DP-iPSCs 三胚層分化的 markers 24
圖九、BEST-iPSCs 的型態及免疫螢光染色 25
圖十、比較 BEST1 與 STXBP2，其 mRNA 與蛋白質表現量的差異 26
圖十一、利用 DPN 以及 DP-Best 處理藥物後，其 BEST1 和 STXBP2 的基因表現量 27
圖十二、DP-Best 處理藥物後，其 BEST1 和 STXBP2 的基因表現量 28
表一、記錄牙齒檢體的年齡、性別與初代培養的日期 29
表二、引子的序列與目標基因的大小 30
表三、免疫螢光染色的初級及次級抗體 31
表四、在 BEST 疾病中，蛋白質表現量明顯低於對照組 (紅色區域)。 32
表五、在 BEST 疾病中，蛋白質表現量明顯高於對照組 (紅色區域)。 33

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