以MMP-3啟動-Luc-GFP報導基因於轉殖大鼠及其初代細胞研究受刺激後表達的時間特性

Temporal Expression of MMP-3 Promotor-Reporter under Stimulation -in Vivo and in Vitro

郭良柔(Liang-Jou Kuo)

指導教授 ： 姚宗珍

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202001328

摘要

MMP-3為一種內切蛋白酶。在不同層面上調控此分子的表現和活性也影響骨質塑形/重塑。在一般生理環境下可以看到此分子表現外，在發炎環境下，例如類風濕性關節炎中，也會因疾病的進展，關節腔或血液內之MMP-3濃度增加。而在特定的機械壓力或是化學刺激之不同實驗模型中，24小時內都可以見到MMP-3明顯的變化。
本實驗使用Mmp-3-luciferase-GFP基因轉殖大鼠，利Luciferase可由冷光偵測觀察MMP-3在受力刺激後時間與空間的相應關係。細胞實驗中也使用關節滑液腔取出培養之纖維母細胞初代細胞，以不同量化報導基因蛋白質的方法，找出此報導基因蛋白質的半生期，以期可以呼應動物實驗中的時間、受力大小、空間分佈等參數之相關性。動物實驗結果發現在十五天的矯正力量施予期間，Luciferase冷光訊號於施力後第一天訊號最為明顯，並於第三天回到與原始基礎訊號相同;即使期間每三天給予低能量雷射照射或是重新給予矯正施力，皆無法促使報導基因再度活化、冷光訊號強度上升。在翻開實驗大鼠臉頰觀察冷光訊號表現時，也可以見到在施予矯正力量後的一天，Luciferase冷光訊號確實出現於受力的第一大臼齒上，並且訊號強度於第一天時強度最強，在放掉力量後三小時開始下降，直至八小時回到與原始基礎訊號相同。而細胞實驗中，給 LPS濃度達 1 ng/ml，刺激八小時後在冷光強度訊號(luciferase assay)可以見到上升趨勢。在西方墨點法中，當LPS濃度為0.5 ng/ml與1 ng/ml，刺激八小時，可以偵測到較為明顯之MMP-3蛋白質表現。
在本實驗動物模型中，可以看到在牙齒受到持續性矯正力或是間歇性矯正力刺激時， MMP-3皆在初期力量刺激時即表現，一天可達高峰，移除力量後很快消失。

關鍵字

基質金屬蛋白酶 ； 基因轉殖大鼠 ； 牙齒矯正 ； 關節滑液纖維母細胞 ； 冷光素酶

並列摘要

MMP-3 is an endoproteinase which the activity is regulated at different levels to influence bone modeling and remodeling. In inflammation, such as rheumatoid arthritis, the increased level of MMP-3 correlated with the severity of diseases. With specific mechanical force or chemical stimulation in different models, MMP-3 significantly and quickly increased within 24 hours.
By observing luciferase driven by Mmp-3-promoter in a transgenic rat model, we studied the temporospacial expression of MMP-3 during orthodontic force stimulation. In vivo, luciferase increased signficantly after 1 day of orthodontic force stimulation then dropped back to baseline on the 3rd day of a fifteen day experiment. Neither low level laser applied every three days, nor orthodontic force reactivation did enhance the signal. Location of luminance was at the molar under orthodontic force for protraction, when cheeks were reflected for a direct view of molars. Once the force removed, the signal decreased and dropped to baseline level after 8 hours. In vitro, isolated synovial fibroblast from transgenic rat was used to measure the rate of protein degradation up on LPS stimulation. A trend of bioluminescence signal was detected after stimulation of LPS 1 ng/ml at 8 hours. In Western blot assay, obvious MMP-3 protein band was shown when stimulation by LPS 1 ng/ml and 0.5 ng/ml at 8 hours.
In our study, we demonstrate that MMP-3 was expressed at the initial phase of mechanical stimulation. Either the continuous force or interrupted force, the bioluminescence signal of luciferase was enhanced after 1 day force application. This rise was quickly tuned down after 3 hours of force termination. Therefore, the regulation of this MMP-3 expression is tightly controlled in a timely fashion. Details of this regulatory mechanism warrant further studies.

並列關鍵字

matrix metalloproteinase ； transgenic rat ； orthodontic force ； synovial fibroblast ； luciferase

參考文獻

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