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系統識別號 U0026-2108201417354900
論文名稱(中文) 比較齒間乳突和邊緣牙齦之間不同的幹細胞標誌表現量
論文名稱(英文) Comparing the expression of different stem cell markers between interdental papilla and marginal gingiva
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 許芳嘉
研究生(英文) Fang-Chia Hsu
電子信箱 s0972976@mail.ncyu.edu.tw
學號 T46011038
學位類別 碩士
語文別 中文
論文頁數 58頁
口試委員 指導教授-袁國
口試委員-陳玉玲
口試委員-吳炳慶
口試委員-許嘉文
中文關鍵字 人類幹細胞  邊緣牙齦組織  齒間乳突組織 
英文關鍵字 Human stem cell  interdental papilla  marginal gingiva 
學科別分類
中文摘要 齒間乳突缺失會造成食物塞入牙縫中造成蛀牙的困擾並且容易造成牙周方面的問題。目前雖然可利用軟、硬組織的移植來解決這種問題,但是因為這個區域的血液供應量有限,手術並不容易成功。有文獻發現到在齒間乳突容易由藥物引發或是遺傳性基因造成牙齦增生的情況,顯示齒間乳突是有容易增生的傾向。因此我們想知道齒間乳突比起邊緣牙齦是否含有較多的幹細胞去分化成特定的細胞,而且以前的文獻也比較少深入去探討兩者之間幹細胞基因的表現差異。所以為了了解齒間乳突和邊緣牙齦之間具有的幹細胞分子表現差異,因此我們從牙冠增長術後取下的26塊健康邊緣牙齦及齒間乳突組織,進行real time PCR array的分析,從中找出兩個組織之間有差異性的基因表現,篩選後找到了12個相對有表現差異性的基因,接下來則對12個基因進行個別real time PCR,西方點墨法和免疫組織化學染色確認real time PCR array中分析出的兩者之間基因的表現差異結果。
從real time PCR實驗結果顯示了有5個基因是符合之前PCR array data,包含MME、SOX1、ALPI、ALDH1A1和ISL1。因此再看蛋白質上的表現,發現在蛋白質的表現與先前的array data有一致性的情況,而在實際組織上的表現,ALDH1A1、MME、ALPI和ISL1做完IHC染色後,進行HistoQuest分析數據,發現在齒間乳突的表現是比邊緣牙齦還多,然而SOX1在齒間乳突中的表現卻比邊緣牙齦還要來的低,但是從結果來看,大致上可以推測齒間乳突是比邊緣牙齦是含有比較多特殊分子的表現,可以使得齒間乳突能夠容易受到刺激生長。未來或許有機會利用組織工程或基因工程重建缺失的齒間乳突。
英文摘要 In previous papers, we can see that there have been comparative studies between interdental papilla and marginal gingival to look at some specific markers. In this study, we have three aims. First, we utilized human stem cell PCR array to explore the gene expression levels between interdental papilla and marginal gingival and also confirmed this phenomenon by using real-time PCR with a different primer. Next, we confirmed the protein levels by western blotting. Finally, We confirmed clinical expression by IHC and utilized HistoQuest to quantify the immunohistochemistry expression. We found that there was a significant difference in the expression levels of SOX1, ALPI, ALDH1A1, ISL1 and MME between interdental papilla and marginal gingiva. Although the IHC expression of SOX1 did not correlate our PCR array data, we still found that ALPI, ALDH1A1, MME and ISL1 were expressed higher in interdental papilla than in the marginal gingival through real-time PCR, western blot and immunohistochemistry. Therefore, our results show that interdental papilla has higher expression levels of stem cell markers than marginal gingiva. Thus, papilla may be more easily stimulated for growth and may be utilized to treat papilla deficiencies.
論文目次 中文摘要-------------------------------------------- I
Abstract------------------------------------------ II
致謝----------------------------------------------- V
目錄----------------------------------------------- VI
表目錄--------------------------------------------- VIII
圖目錄--------------------------------------------- IX
英文縮寫檢索表--------------------------------------- X
第一章 緒論----------------------------------------- 1
一、牙齦組織----------------------------------------- 1
1. 牙齦萎縮(Gingival recession)--------------------- 3
2. 牙齦增生(gingival overgrowth)-------------------- 4
二、幹細胞標誌(stem cell marker)--------------------- 4
1. 膜金屬內肽酶(membrane metallo-endopeptidase, MME)- 5
2. SOX1(SRY (sex determining region Y)-box 1)------ 6
3. 鹼性磷酸腸道酶(Alkaline phosphatase, intestinal, ALPI) 6
4. 醛脫氫酶(Aldehyde dehydrogenase 1 family, member A1, ALDH1A1)------------------------------------------- 7
5. 胰島素基因增強子蛋白(ISL LIM homeobox 1)------------ 7
三、研究動機------------------------------------------ 7
第二章 實驗材料與方法---------------------------------- 9
一、人類牙齦組織來源及保存------------------------------ 9
二、萃取RNA (RNA extraction)------------------------- 9
三、洋菜膠體電泳(Agarose gel electrophoresis)--------- 12
四、反轉錄PCR (RT- PCR)------------------------------ 13
五、Human Stem Cell Polymerase Chain Reaction(PCR) assay 14
六、即時定量聚合酶連鎖反應(real-time PCR)--------------- 15
七、組織蛋白質萃取(Protein extraction)---------------- 16
八、蛋白質濃度測定(Protein assay)--------------------- 17
九、SDS-PAGE 蛋白質電泳(SDS-PAGE protein electrophoresis) 18
十、西方點墨法(Western blot)-------------------------- 19
十一、免疫組織化學染色法(Immunohistochemistry, IHC)----- 21
十二、影像處理---------------------------------------- 24
十三、統計方法---------------------------------------- 24
十四、儀器設備、試劑藥品、套裝試驗組、抗體、耗材------------- 25
第三章 實驗結果--------------------------------------- 30
一、人類牙齦組織之幹細胞標誌(stem cell marker)表現分析---- 30
二、Human stem cell PCR array分析後有表現差異的基因----- 30
三、利用Real-time PCR去確認從Human stem cell PCR array中找出有差異性的幹細胞標誌-------------------------------------- 31
四、比較ALDH1A1、ALPI、ISL1、MME、SOX1在interdental papilla和marginal gingiva protein的表現量--------------------- 32
五、證明ALDH1A1、ALPI、ISL1、MME、SOX1在interdental papilla組織中的表現量是高於在marginal gingiva組織------------------ 32
第四章 實驗討論--------------------------------------- 34
第五章 結論------------------------------------------- 38
參考文獻---------------------------------------------- 39
結果(附表)-------------------------------------------- 43
結果(附圖)-------------------------------------------- 46
附錄------------------------------------------------- 57
自述------------------------------------------------- 58
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