『基質金屬蛋白酶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

Abstract

基質金屬蛋白酶3 (MMP-3) 參與許多生理機能運作，細胞受矯正機械力刺激後可對MMP-3進行調控，但此密切關係仍有許多未明朗處。為進一步了解MMP-3受機械力調控的機制，本實驗利用先前複製出的人類MMP-3基因啟動子， 先建立『基質金屬蛋白酶3基因啟動子- 綠螢光蛋白』之基因轉殖小鼠，進入 in vivo實驗的範疇。基因轉殖小鼠建立後的實驗，大致分為三部分，1)機械力刺激：於小鼠口內牙齒施予矯正力，了解MMP-3在矯正牙齒移動時的表現位置，並嘗試以不同時間點分析螢光強度是否變化；2)發炎反應刺激： 進行膠原蛋白或佐劑誘導之關節炎 (collagen or adjuvant induced arthritis model)，誘發小鼠產生關節炎，觀察小鼠腳踝關節有無綠螢光，即MMP-3產生的情形；3)修復過程：製造小鼠背部傷口，合併尾巴根部在膠原蛋白誘導之關節炎實驗中所產生的傷口，觀察傷口表層有無綠螢光反應。本研究結果顯示MMP-3在矯正牙齒周遭組織分布情況，不論門齒或臼齒，主要在張力側出現綠螢光，且位在牙周韌帶與骨頭交界處，撐開的正中顎骨縫也可見綠螢光，且第四天的螢光亮度明顯高於第三天，最弱反應的則為第七天。而本實驗中的基因轉殖鼠對膠原蛋白誘導之關節炎反應較低，觀察到第50天都未見到關節腫脹反應，另外由佐劑誘導的組別，可成功誘發關節腫脹，但切片觀察皆未見到綠螢光。尾巴傷口觀察發現，第28天有一隻小鼠在活體照射UV燈下，發現螢光反應，卸下21天的尾巴傷口切片在螢光顯微鏡下卻未能見到綠螢光。 因此矯正牙齒移動與傷口癒合在不同時間點，所產生的MMP3綠螢光之亮度也不同，有待後續實驗進一步找出最佳時間點 。

Matrix metalloproteinase-3 (MMP-3) which degrades proteoglycans, fibronectin, laminins and gelatin in extracelluar matrix, participates in multiple physiological functions. Previously, mechanical force stimulation was found to up-regulate MMP-3 expression. 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 upregulation in mouse osteoblasts, and then created “mmp-3 promoter - green fluorescent protein” transgenic mouse model for in vivo test. After transgenic mice lines were established, three types of stimulation were tested for GFP expression. 1) mechanical force stimulation: applying orthodontic force on the teeth of transgenic mouse for various periods of time to detect the temporal and spatial distributions of GFP in periodontal tissue; 2) inflammatory stimulation: using collagen or adjuvant-induced arthritis (CIA) model to see if the GFP can accumulate at the ankle joints; 3) wound repair process: observing the intentionally wounded back skin and also the injection site wound in CIA model on the tail roots. The results showed the distribution of MMP-3 in periodontal tissue either at incisors or molars, the GF appeared mainly at the tension side, and located at the junction of periodontal ligament and bones, also in the distracted mid-palatal suture. The GF was brightest on the fourth day compared to the intensity on the third day which still with significant signal. But on the seventh day the signal dropped down significantly. To our surprise, CIA could not be induced in our transgenic lines, after 50 days of observation, there was no joint swelling after collagen injection. In another group of adjuvant-induced arthritis, joint swelling could be successfully induced, but no GF was detected histologically. Interestingly, strong GF on a 28-day tail wound when UV light shone on the animal was noticed in one incident, but we failed to detect GF in 21-day tail wound histological sections under fluorescence microscope. Therefore, we conclude that the mmp-3 GFP transgenic lines can successfully be used in orthodontic tooth movement and wound healing process models since localized signals with different intensity at various time points were detected. Further experiments should be performed to optimize the methods for detecting the GF signals in these various models for tracking its real time expression.