臺灣博碩士論文加值系統

成人幹細胞，存在於成體特定的組織中，具有由幹原細胞形成前驅細胞，分化成具特定功能細胞的能力。牙齒幹細胞也是其中之一，牙齒幹細胞有很多種，其中有恆牙牙髓幹細胞以及牙周韌帶幹細胞。在先前的研究中指出，牙髓幹細胞能分化成牙本質母細胞，並促進牙本質的再生作用。而牙周韌帶幹細胞可能主要源自血管的周圍。在其他文獻中，使用牙髓幹細胞以及牙周韌帶幹細胞來比較細胞表面的基因標記，從中發現了兩者細胞皆含有(Stro-1, CD73, CD44, CD146, CD105, CD106, CD29)等標記。綜合以上的文獻發現，目前尚無深入探討幹細胞基因的表現性差異。因此在本篇研究中，首先取用二十顆18-25歲的第三大臼齒，取出牙髓組織以及牙周韌帶組織，並利用人類幹細胞的聚合酶連鎖反應的微陣列去比較兩個組織之間的基因表現差異。在以牙周韌帶組織為控制組比較時發現，CD44 (CD44 molecule)、ACAN (Aggrecan)基因的表現量較牙髓組織低。CDH1 (Cadherin 1, type 1)、COL1A1 (Collagen, type 1, alpha 1)、NUMB (Numb homolog)、BTRC (Beta-transducin repeat containing)、基因的表現量較牙髓組織高。我們也再次設計不同的引子做定量聚合酶連鎖反應去確認來證實，我們也利用了西方墨點法去觀察蛋白質的表現量，從實驗結果得知牙髓組織的ACAN及CD44之蛋白質表現量較牙周韌帶組織高，而在CDH1、NUMB、BTRC、COL1A1的蛋白質表現量較牙周韌帶組織低。接下來我們使用免疫組織化學染色來比較牙髓組織以及牙周韌帶組織中幹細胞的基因表現，我們觀察到在臨床的檢體中幹細胞基因表現的差異確實也與前面有一致的結果。以上結果可證明牙髓組織及牙周韌帶組織幹細胞某些相關基因表現具有差異。此研究或許有助於未來在牙科再生醫學的應用。

In previous studies, we can see that few studies have discussed that difference exist between stem cell markers. In this study, we have three objective. First, we compare dental pulp and periodontal ligament and look at the difference between human stem cell associated genes. Next, we look for markers that are significantly different between these two types of cells. And look at the function of those stem cell associated genes. Last, we explore the clinical expression between dental pulp and periodontal ligament. Our analysis indicated that different tissues had different expression levels of specific stem cell associated genes. We founded that there was a significant difference in the expression levels of ACAN, CD44, COL1A1, NUMB, BTRC, and CDH1 between pulp and PDL. We found that these genes are not entirely correlated with human stem cells. However, we observed that there are many different genes between pulp and PDL, and the results could help with understanding of the tissue biology. The molecular mechanisms correleated witht these genes can be looked at in greater depth in future expreiments.

中文摘要 I
英文延伸摘要(EXTENDED ABSTRACT) II
致謝 V
目錄 VI
表目錄 IX
圖目錄 X
英文縮寫檢索表 XI
第一章 緒論 1
一. 牙齒生成與形態學概論 1
1. 牙齒功能與組成 1
2. 牙齒周圍組織 2
二. 幹細胞概論(Stem cells) 2
1. 幹細胞的分類 2
三. 牙齒幹細胞(Dental stem cells) 4
1. 牙齒幹細胞分類 4
2. 牙齒幹細胞標誌(Dental stem cell markers) 4
四. 牙齒幹細胞的優勢與未來 5
1. 牙髓幹細胞的發展與應用(DPSCs) 5
2. 牙周韌帶幹細胞的發展與應用(PDLSCS) 5
五. 研究動機 6
第二章 實驗材料與方法 7
一. 成人牙齒之收集來源 7
二. 分離成人牙髓組織以及牙周韌帶組織 7
A. 分離成人牙髓組織 7
B. 分離成人牙周韌帶組織 7
三. 組織萃取RNA(RNA Extraction) 8
四. 洋菜膠體電泳(agarose gel electrophoresis) 9

五. 反轉錄PCR(RT-PCR) 10
六. 人類相關幹細胞PCR Array(Human stem cell PCR array) 11
七. 即時定量聚合酶連鎖反應(Real-time PCR) 12
八. 蛋白質濃度測定(Protein assay) 13
九. SDS-PAGE 蛋白質電泳(SDS-PAGE Protein electrophoresis) 14
十. 西方點墨法(Western blot) 15
十一. 組織蛋白質萃取(Protein extraction) 17
十二. 免疫組織化學染色(Immunohistochemistry, IHC) 18
十三. 統計分析(Statistic methods) 21
十四. 儀器設備 試劑藥品 套裝實驗組 抗體 耗材 21
第三章 實驗結果 27
一. 組織檢體的取得、分離、萃取 27
二. 利用人類幹細胞(Human stem cell) PCR array 比較兩者有差異的基因 27
三. 利用Real-time PCR再次確認Human stem cell PCR array 有差異的基因 28
四. 分析比較ACAN、CD44、COL1A1、NUMB、BTRC、CDH1在pulp和PDL的蛋白質(Protein)表現量 29
五. 證明確認ACAN、CD44、COL1A1、NUMB、BTRC、CDH1在臨床人類組織的表現 29
六. 證明確認ACAN、CD44、COL1A1、NUMB、BTRC、CDH1在FVB老鼠組織的表現 30
第四章 實驗討論 31
第五章 結論 36
參考文獻 37
附錄 62
自述 63


表目錄
表 一 41
表 二 42
表 三 43
表 四 45

 
圖目錄

圖 一 45
圖 二 46
圖 三 47
圖 四 48
圖 五 49
圖 六 50
圖 七 51
圖 八 52
圖 九 53
圖 十 54
圖 十一 55
圖 十二 56
圖 十三 57
圖 十四 58
圖 十五 59
圖 十六 60
圖 十七 61

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