週期性張力與壓力刺激對細胞外基質金屬蛋白酶-3基因啟動子的調控

Abstract

機械力量的刺激對骨骼在生長發育中的重塑扮演重要的角色，但力量刺激對細胞內基因變化所受到的調控，目前仍然不清楚。之前的研究已利用基因微陣列分析，篩選出43 個基因在MG-63 類成骨細胞受壓力刺激後有差異性的表現，其中細胞外間質代謝酶三(Matrix Metalloproteinase 3, MMP-3)受壓力刺激後在RNA層次（上升2.3 倍）及Protein 層次上會有上升的情形。本實驗先從人類細胞設計 兩段帶有酵素切位的MMP-3 promoter primer，利用聚合酶連鎖反應（PCR）夾出約3.2K bp 的片段後，接到pGL3-basic vector 及pEGFP-1 vector 上，純化出轉殖成功的pGL3 basic-MMP-3 及pEGFP-1-MMP-3 plasmid。將上述plasmid 轉染到MG-63 類成骨細胞內，之後進行張力及壓力實驗。當帶有pGL3 basic-MMP-3 及pEGFP-1-MMP-3 plasmid 的細胞受到張力刺激，分別利用冷光分析儀讀取冷光數 值及倒立式螢光顯微鏡直接觀察後，發現MMP-3 promoter 活性調控受到張力刺激會有微量下降的情形。在西方墨點法分析中亦可見細胞內MMP-3 protein 受張力刺激後，有微量的下降。而在受到壓力的刺激後，利用冷光分析儀讀取冷光數值及西方墨點法分析細胞內MMP-3 protein 表現量，發現會有明顯的上升。因此，週期性張力及壓力刺激在對MMP-3 promoter 基因調控，蛋白質的表現量反應不同，前者為正向調控，後者為負向調控。

It is widely accepted that mechanical loading is necessary to construct the architecture of bone and to maintain bone mass. Mechanical strain plays an important role in bone remodeling during growth and development. However, the cellular gene expression that controls the tissue response to mechanical force remains elusive. Previous experiments detected increased expression of matrix metalloproteinase-3 (mmp-3) when cyclic compression force was applied to MG-63 osteoblast-like cell in 3D collagen gel. Here, I cloned a 3.2 K-mmp-3 promoter fragment from human normal and HeLa cell by polymerase chain reaction and inserted this DNA into pGL3-Basic and pEGFP-1 vectors. Purified promoter-reporter constructs were transfected into MG-63 cells and cells were then processed through cyclic tension and compression stimulation for one day. The results indicate that the MMP-3 promoter activity was slightly down regulated by cyclic tension stimulation. However, promoter activity was up regulated by cyclic compression. Hence, mmp-3 promoter activity could be differentially regulated when osteoblasts experience different patterns of mechanical stimulation.