骨形态发生蛋白2/Osterix信号通路调控的前成骨细胞分化

摘要

BACKGROUND:Bone formation and development are reported to be regulated by bone morphogenetic protein2(BMP2)-induced Osterix expression. OBJECTIVE:To investigate the regulatory effect of BMP2/Osterix signaling pathway on differentiation of preosteoblasts in mice. METHODS:mRNA and protein expression of Osterix wasdetermined by real-time RT-PCR and western blot assay, respectively at various time points after mouse preosteoblasts were treated with BMP2. pcDNA3.1/myc-Osterix eukaryotic expression vector was constructed and transducted into preosteoblasts, and then mRNA expression of alkaline phosphatase, bone sialoprotein, and matrix extracelular phosphoglycoprotein wasdetected by real-time RT-PCR after transduction and BMP2 treatment. RESULTSANDCONCLUSION:Osterix mRNA expression was up-regulated when treated with BMP2 in mouse preosteoblasts, and reached the peak at 24 hours. In addition, the protein expression of Osterix was increased after BMP2 treatment. Alkaline phosphatase, bone sialoprotein, and matrix extracelular phosphoglycoprotein mRNA expression wasup-regulated after transfection of mouse preosteoblasts with pcDNA3.1/myc-Osterix eukaryotic expression vector and BMP2 treatment. Our results indicate that BMP2 regulates the synthesis of genetic markers of osteogenesis,such asalkaline phosphatase,matrix extracelular phosphoglycoproteinviaup-regulating Osterix expression in mouse preosteoblasts, suggesting BMP2/Osterix signaling pathway plays a critical role in bone development.%背景：研究发现Osterix基因的表达能够受到骨形态发生蛋白2信号通路的调控，从而调节骨的形成发育。目的：分析骨形态发生蛋白2/Osterix信号通路对小鼠前成骨细胞分化的调控作用。方法：将骨形态发生蛋白2作用于前成骨细胞，利用实时定量RT-PCR法及免疫印迹法检测不同时间段Osterix mRNA和蛋白的表达水平；构建pcDNA3.1/myc-Osterix真核表达载体，并转染至前成骨细胞，利用实时定量RT-PCR法检测转染Osterix真核表达载体和骨形态发生蛋白2作用后碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白(MEPE)的mRNA表达水平。结果与结论：①实时定量RT-PCR法检测结果表明骨形态发生蛋白2上调Osterix mRNA在小鼠前成骨细胞中的表达水平，并在24 h时上调作用最为显著；②免疫印迹法检测骨形态发生蛋白2显著上调Osterix 蛋白的表达水平；③成功构建了 pcDNA3.1/myc-Osterix 真核表达载体，在转染小鼠前成骨细胞和以骨形态发生蛋白2作用后检测到碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白的mRNA表达显著增强；④结果说明，骨形态发生蛋白2通过上调小鼠前成骨细胞中Osterix的表达来调控碱性磷酸酶、细胞外基质磷酸化糖蛋白等成骨标志基因的合成，表明骨形态发生蛋白2/Osterix这一信号通路在骨发育过程中具有重要作用。