背景：细胞外基质磷酸化糖蛋白基因在骨的矿化和吸收、成骨细胞与破骨细胞的平衡中起重要的作用，研究细胞外基质磷酸化糖蛋白的功能及其调控机制可为骨质疏松症的治疗提供新的思路。 目的：分析转录因子Runx2在小鼠前成骨细胞中对细胞外基质磷酸化糖蛋白基因启动子的调控作用，进而初步研究转录因子Runx2在骨形成发育过程中的作用。 方法：首先根据Genbank中Runx2的基因序列构建Runx2真核表达载体；然后利用双荧光素酶基因检测报告系统分析Runx2对不同长度的细胞外基质磷酸化糖蛋白基因启动子转录活性的影响，以确定Runx2有明显作用的启动子区段，分析3种MAPK信号通路抑制剂调控Runx2对细胞外基质磷酸化糖蛋白基因启动子转录活性的影响；最后利用定时定量RT-PCR法分析Runx2对细胞外基质磷酸化糖蛋白基因启动子表达活性的影响。结果与结论：成功构建Runx2真核表达载体；双荧光素酶基因检测报告系统分析显示Runx2能够上调细胞外基质磷酸化糖蛋白基因启动子在前成骨细胞中的转录活性，在(-300-+66)366 bp片段区域内上调效果较为显著，Runx2可通过激活MEK激酶MAPK通路上调细胞外基质磷酸化糖蛋白启动子活性；定时定量RT-PCR法检测再次验证Runx2上调细胞外基质磷酸化糖蛋白基因启动子的表达水平。提示转录因子Runx2可以通过MEK激酶MAPK信号通路对细胞外基质磷酸化糖蛋白基因表达进行调节，为探讨细胞外基质磷酸化糖蛋白在骨形成发育过程中的意义奠定基础。%BACKGROUND:Matrix extracel ular phosphoglycoprotein phosphorylated extracel ular matrix glycoprotein (MEPE) gene plays an important role in bone mineralization and absorption as wel as the balance of osteoblasts and osteoclasts. Studies on the function and regulatory mechanism of MEPE can provide new ideas for the treatment of osteoporosis. OBJECTIVE:To analyze the regulatory effects of transcription factor Runx2 on MEPE promoter in mouse preosteoblasts, thereby preliminarily studying the Runx2 effects in the process of bone formation and development. METHODS:First of al , the Runx2 eukaryotic expression vector was built according to the gene sequence of Runx2 in Genebank;then the dual luciferase reporter assay was employed to analyze the effects of Runx2 on transcription activity of MEPE promoters with different lengths in order to determine the promoter region in which Runx2 has significant effect. Afterwards, the effects of Runx2 on transcipition activity of MEPE gene promoter which induced by three MAPK signaling pathway inhibitors were investigated. Final y, real-time PCR was used to analyze the expression activity of MEPE gene promoter regulated by Runx2. RESULTS AND CONCLUSION:We successful y constructed the Runx2 eukaryotic expression vector. Dual luciferase reporter assay showed that Runx2 could increase the transcription activity of MEPE gene promoter in preosteoblasts, and the fragment area in which Runx2 exhibited the more significant up-regulatory effectiveness was (-300 to+66)366 bp. Runx2 could increase the transcription activity of MEPE gene promoter by activating the MAPK single pathway. The real-time PCR verified that Runx2 increased the expression activity of MEPE gene promoter. These findings indicate that Runx2 can regulate the express of MEPE gene promoter by the MAPK single pathway, in order to build the basis for exploring the process of bone formation and development.
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