週期性壓力刺激對於細胞外基質金屬蛋白酶-3基因啟動子在老鼠類骨母細胞內的調控

近年來，機械力量刺激對於骨質重塑的調控機制在矯正學上受到重視。而根據文獻研究，在矯正治療受壓力區的骨質吸收與基質金屬蛋白酶家族(MMPs)中的基質金屬蛋白酶-3(MMP-3)有密切相關。有些研究文獻也認為基質金屬蛋白酶-3 (MMP-3)基因表現調控可受到機械壓力刺激的影響，和關節及受壓的椎間盤的骨吸收作用有相關。本研究以老鼠類成骨細胞株MC3T3-E1細胞作為實驗對象，培養於3D膠原蛋白凝集體中，模擬生理環境，施予細胞1%週期性壓力刺激4、8、24小時後，以即時定量聚合酶鏈鎖反應(Real-time PCR)分析，發現MMP-3基因的表現隨著壓力時間有所變化，一開始雖略微下降，後來刺激到達24小時，MMP-3 mRNA表現明顯增加，平均提升4.204倍。並且將人類MMP-3基因啟動子(MMP-3promoter)轉染到老鼠MC3T3-E1細胞中，觀察壓力刺激對於人類MMP-3 promoter活性表現的影響，結果也發現接受1%週期性壓力刺激24小時後，人類MMP-3 promoter冷光活性平均增加4.62倍。為了更近一步探討老鼠類成骨細胞表現MMP-3的可能訊息傳導路徑，本實驗於MC3T3-E1細胞受壓1小時前加入5種inhibitor，分別為加入5種inhibirors，分別是MEK1/2 inhibitor (U0126)， p38 inhibitor (SB203580)，JNK II inhibitor (420128)，NF-κB inhibitor (BAY 11-7082)，PI3-K inhibitor (LY 29400)，並以冷光分析探討MMP-3可能的機械力刺激訊息傳導路徑，結果發現加入p38 inhibitor (SB203580)和PI3-K inhibitor (LY 29400)後，MMP-3 promoter活性表現有向下調控的現象，因此可推測人類MMP-3 promoter於老鼠MC3T3-E1細胞中，接受壓力刺激後其活性向上調節的現象可能經由p38 pathway、以及PI3-Kinase pathway來進行訊息傳導。

關鍵字

基質金屬蛋白酶機械力量刺激； MC3T3-E1

並列摘要

The mechanism of bone remodeling by mechanical stimulation became an important issue in orthodontics recently. Previously, bone resorption in compression area during orthodontic treatment was found to be closely related to the expression of matrix metalloproteinases-3 (MMP-3), one of the members of matrix metalloproteinases family (MMPs). Also, the regulation of MMP-3 has been reported to be modified by mechanical compression in other portions of body, and involve bone resorption in arthritic joints and compressed intervertebrate discs. In order to verify how the mechanical compression may affect the MMP-3 gene expression, 1% cyclic compression force for 4, 8, 24 hours was applied to mouse osteoblast-like MC3T3-E1 cells grown in a 3D collagen gel to mimic the physiologic environment. The result of real-time PCR showed MMP-3 mRNA expression changed in a time-dependent manner, mild down-regulation at early times, but increased significantlty after 24 hour of compression, which was up-regulated for average 4.204-fold. In addition, luciferase activity of human MMP-3 promoter was also up-regulated average 4.62-fold in mouse MC3T3-E1 cells after 1%, 24 hours cyclic compression force. For identifying possible signaling pathways of MMP-3 promoter regulation, 5 inhibitors including MEK1/2 inhibitor (U0126)， p38 inhibitor (SB203580)，JNK II inhibitor (420128)，NF-κB inhibitor (BAY 11-7082)，PI3-K inhibitor (LY 29400) were tested. The dual luciferase ananlysis showed that p38 inhibitor (SB203580)和PI3-K inhibitor (LY 29400) both had down-regulated MMP-3 promoter. Thus we concluded that MMP-3 gene expression in mouse MC3T3-E1 cells was up-regulated by 1%, 24 hours cyclic compression, and so was the transfected human MMP-3 promoter. And the p38和PI3-K pathways possibly involved for the mechanical stimulated expresssion of MMP-3 promoter.

並列關鍵字

matrix metalloproteinase ； mechanical stress ； MC3T3-E1 osteoblast

參考文獻

汪文琲 (2006) 細胞間質代謝酶在矯正治療中的角色. 國立台灣大學醫學院臨床牙醫學研究所碩士論文.

翁竹音 (2005) 週期性壓力刺激對類成骨母細胞基因表現的影響. 國立台灣大學醫學院臨床牙醫學研究所碩士論文.

Abreu, J. R., D. de Launay, M. E. Sanders, A. M. Grabiec, M. G. van de Sande, P. P. Tak and K. A. Reedquist (2009) The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue. Arthritis Res Ther. 11, R121.

Adya, R., B. K. Tan, A. Punn, J. Chen and H. S. Randeva (2008) Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res. 78, 356-65.

Akhouayri, O., M. H. Lafage-Proust, A. Rattner, N. Laroche, A. Caillot-Augusseau, C. Alexandre and L. Vico (1999) Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels. J Cell Biochem. 76, 217-30.

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週期性壓力刺激對於細胞外基質金屬蛋白酶-3基因啟動子在老鼠類骨母細胞內的調控

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