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研究生:林昇進
研究生(外文):Sheng-Chin Lin
論文名稱:『基因金屬蛋白-3基因啟動子-綠螢光蛋白』之基因轉殖小鼠經矯正施力後之螢光表現
論文名稱(外文):The induction of fluorescence by orthodontic force in MMP-3 promoter-GFP transgenic mice
指導教授:姚宗珍
指導教授(外文):Chung-Chen Yao
口試委員:張百恩劉佳觀
口試委員(外文):Bei-En ChangJia-Kuang Liu
口試日期:2014-07-18
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
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:2014
畢業學年度:102
語文別:中文
論文頁數:65
中文關鍵詞:基質金屬蛋白&;#37238;基因轉殖小鼠矯正牙齒移動
外文關鍵詞:Matrix metalloproteinaseTransgenic miceOrthodontic tooth movement
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基質金屬蛋白&;#37238;-3(MMP-3)和許多生理機能運作以及病理機制相關,在矯正牙科治療中也在骨質重塑的過程,扮演重要的角色。然而此密切關係仍待釐清。本實驗室利用先前複製出的人類MMP-3基因啟動子,建立了『基質金屬蛋白&;#37238;-3基因啟動子-綠螢光蛋白』之基因轉殖小鼠,希望可以藉由in vivo的方式,透過綠螢光觀察MMP-3在牙齒矯正受力後的表現,代表MMP-3在矯正中活化的區域與時間。本研究利用此基因轉殖小鼠進行實驗包括:(一)觀察施予矯正力量後綠螢光出現的位置,並試著找出與矯正施力方向的關聯性;(二)觀察力量刺激下經過不同時間後綠螢光的強度變化以及力量消失後螢光可維持的時間,進而找出MMP-3表現強度的時間軸;(三)利用免疫組織化學染色的方式直接染出MMP-3和GFP蛋白質的空間分佈並找出與螢光出現位置的關聯性。本研究結果顯示未脫鈣樹脂切片綠螢光的表現主要靠近張力側的齒槽骨和牙周韌帶交界,而受壓力側明顯則較觀察不到螢光。螢光強度隨著矯正施力的時間不同而有所變化:在施力一天至兩天綠螢光達到最大亮度,之後逐漸變暗;而在施力一天再將裝置移除後,大約在3~4小時時綠螢光開始變得不明顯。免疫組織化學染色的結果顯示經矯正施力後MMP-3染色範圍較GFP廣,在施力三天時仍可觀察到MMP-3表現,而GFP已變得較為不明顯。由基因轉殖小鼠我們可以觀察到綠螢光在矯正施力後不同時間點與空間的變化,後續的研究將更準確且有效率地找到MMP-3表現與矯正施力的時間軸,以及用不同施力頻率或方式,去尋求最佳刺激參數。

Matrix metalloproteinase-3 (MMP-3) degrades a number of extracellular matrix proteins and participates in multiple physiological functions and pathological processes. Mechanical force stimulation has been found to up-regulate MMP-3 expression in orthodontic tooth movement (OTM). To further understand the mechanism of regulation of MMP-3 by the mechanical force, we cloned the human MMP-3 gene promoter and identified its up-regulation in mouse osteoblasts, and then generated a MMP-3 promoter-green fluorescent protein (GFP) transgenic mouse model for in vivo test. After MMP-3-GFP transgenic mice lines were established, we applied mechanic force on teeth and addressed the following: 1) the location of GFP expression and its relationship with the direction of tooth movement; 2) the timeline of GFP expression after mechanical stimulation; and 3) the spatial correlation of MMP-3 and GFP by using the immunohistochemistry (IHC).
The results showed that GFP appeared mainly at the tension side and located at the junction of periodontal ligaments and alveolar bone, while it was not found at the pressure side. The intensity of GFP reached its peak after continuous mechanical stimulation for 1 to 2 days in the incisor expansion model. After we applied force for 1 day and then removed the appliance for 3 to 4 hours, the fluorescence was no longer detectable. IHC further improved the understanding of the spatial distribution of MMP-3 and GFP: MMP-3 protein was stained longer and more broadly than GFP since GFP could hardly be observed after 3 days of mechanical stimulation while MMP-3 still existed. Therefore, we conclude that the MMP-3-GFP transgenic mice can be used in OTM study since signals were triggered in a site- and time-specific manner. Further experiment should improve the accuracy and efficiency for detecting the expression of MMP-3 via reporter gene expression and possibly to find optimal mechanical parameter to stimulate the level of MMP-3.



口試委員會審定書………………………………………i
誌謝………………………………………………………ii
中文摘要 關鍵字………………………………………iii
英文摘要 關鍵字………………………………………iv
目錄………………………………………………………v
圖次和表次目錄…………………………………………vii
第一章 引言……………………………………………1
1.1 牙齒矯正基本原理……………………………1
1.2 MMPs 和 MMP-3 簡介…………………………1
1.3 機械力量刺激對細胞表現MMP-3之影響…………3
1.4 MMP-3於矯正施力後不同時間的表現變化………4
第二章 實驗目的………………………………………6
第三章 實驗材料及方法………………………………7
3.1 基因轉殖小鼠的建立過程介紹………………7
3.2 基因轉殖小鼠之基因鑑定(genotyping)…………7
3.2.1 基因萃取 (Genomic DNA extraction)……7
3.2.2 聚合酵素鎖鏈反應 ……………………8
3.3 動物實驗………………………………………………8
3.3.1 飼養環境………………………………………8
3.3.2 實驗動物選取…………………………………8
3.3.3 小鼠麻醉………………………………………8
3.3.4 矯正裝置放置…………………………………9
3.3.5 動物犧牲………………………………………10
3.3.6 切片製作和觀察………………………………10
第四章 實驗結果…………………………………………………12
4.1 基因轉殖小鼠的篩選(genotyping)…………………12
4.2 樹脂切片的綠螢光觀察…………………………………12
4.2.1 門齒撐寬………………………………………12
4.2.2 門牙壓窄………………………………………13
4.2.3 臼齒撐寬………………………………………13
4.2.4 牙髓暴露………………………………………13
4.3 冷凍切片的綠螢光觀察…………………………………13
4.4 石蠟切片的免疫組織化學染色…………………………13
4.4.1 控制組未施予矯正力量………………………13
4.4.2 門齒撐寬………………………………………13
4.4.3 門齒壓窄……………………………………14
4.4.4 臼齒撐寬……………………………………14
4.4.5 其他周邊組織的染色情形…………………14
第五章 討論………………………………………………………16
5.1 綠螢光特性介紹、選擇和其限制………………………16
5.2 矯正牙齒移動後的綠螢光分布…………………………17
5.2.1 控制組………………………………………………17
5.2.2 門齒撐寬…………………………………………18
5.2.3 門齒壓窄…………………………………………19
5.2.4 臼齒撐寬…………………………………………19
5.3 門齒撐寬於不同施力時間下綠螢光的表現情形………20
5.4 石蠟切片的免疫組織化學染色…………………………22
5.4.1 控制組…………………………………………22
5.4.2 門齒撐寬………………………………………22
5.4.3 門齒壓窄………………………………………23
5.4.4 臼齒撐寬………………………………………23
5.4.5 其他周邊組織的綠螢光表現和IHC染色結果…23
第六章 結論………………………………………………………26
第七章 未來研究方向……………………………………………28
附表和附圖…………………………………………………………29
參考文獻……………………………………………………………63


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