Construction and Application of 5’ Regulatory Sequences of Murine Matrix Metalloproteinase 9 (MMP-9) Gene Expression Vector In Vitro

• Main Authors: Jau-Shiun Yu, 余昭勳
• Other Authors: Hsi-Tien Wu
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/01413806676623119230

碩士 === 國立嘉義大學 === 生物農業科技學系碩士班(Institute of Agricultural === 98 === Matrix metalloproteinase 9 (MMP-9) belongs to the family of zinc-dependent matrix metalloproteinases (MMPs), is capable of degrading components of extracellular matrix (ECM) and plays an important role in physiological and pathological processes. Tumor necrosis factor-α (TNF-α) is considered a cytokine with pleiotropic biologic capabilities, and led to a serious of cardiovascular diseases such as atherosclerosis, when it released and stimulated MMP-9 expression and activation. According to the clinical studies, plasma MMP-9 concentration could be used as a novel predictor of the risk of cardiovascular diseases. Because of MMP-9 expression had been largely ascribed to activate gene transcription, we constructed the 2.2 kb and 5.1 kb region of 5’ regulatory sequences of mouse MMP-9 gene, respectively, and studied the transgene and endogenous MMP-9 expression by TNF-α induced in H9c2(2-1) cardiomyoblast. Our results from RT-PCR and gelatin zymography at H9c2(2-1) showed, that induction of MMP-9 expression and activation were dependent on the concentration (0, 10, 20, 50 and 100 ng/mL) and incubation time (24, 48, 72 and 96 hrs) of TNF-α. The 2.2 kb of 5’ regulatory sequence of mouse MMP-9 gene expression was stronger than 5.1 kb in quantity; the same result of endogenous MMP-9 gene expression could be seen when H9c2(2-1) treated with TNF-α. However, the expression sensitivity of 5.1 kb was better than 2.2 kb on the TNF-α (10 ng/mL) treatment. The effect of TNF-α-induced MMP-9 expression under curcumin (20 μM), genistein (20 μM) and coenzyme Q10 (20 μM) treatment was observed. Curcumin and genistein, but not coenzyme Q10, significantly suppressed TNF-α (10 ng/mL) -induced 2.2 kb of mouse MMP-9 transgene expression and endogenous MMP-9 activation (P < 0.05). The inhibitory effect of TNF-α-induced expression by curcumin and coenzyme Q10 coordinately or curcumin, genistein and coenzyme Q10 coordinately was better than curcumin alone, but with no obvious differences. These results suggested that the 2.2 kb of 5’ regulatory sequences of mouse MMP-9 gene with high sensitivity of luciferase reporter gene not only mirrored endogenous MMP-9 expression, but also applied to multi-purposes in the future, such as the in vitro 5’ regulatory sequences of MMP-9 gene expression analysis system, the studied of 5’ regulatory sequences of MMP-9 gene regulation by transgenic animal model or gene therapy applied in heart failure disease.