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目的:研究Runx1在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)成骨分化中的作用。方法:用BMP9腺病毒感染MEFs细胞,利用RT-PCR和Western blot分别在mRNA和蛋白质水平检测Runx1的内源性表达;构建过表达Runx1的重组腺病毒Ad-Runx1,并在mRNA和蛋白质水平验证Ad-Runx1的效果;用Ad-Runx1和BMP9条件培养基共处理MEFs,检测成骨早期指标碱性磷酸酶(ALP)染色和活性,茜素红S染色检测成骨晚期指标钙盐沉积;RT-PCR和Western blot分别检测成骨关键转录因子Runx2在mRNA和蛋白质水平的变化。结果:Ad-BMP9处理MEFs细胞后,可使Runx1在mRNA和蛋白质水平表达上调;构建的Ad-Runx1处理MEFs后,可使Runx1在mRNA和蛋白质水平表达上调;Ad-Runx1处理BMP9诱导的MEFs细胞后,增强了ALP活性和钙盐沉积以及Runx2在mRNA和蛋白质水平的表达。结论:Runx1可以促进BMP9诱导的间充质干细胞MEFs的成骨分化。
AIM: To investigate the role of Runx1 in osteogenic differentiation of mouse embryonic fibroblasts (MEFs) induced by bone morphogenetic proteins 9 (BMP9). METHODS: MEFs were infected with BMP9 adenovirus and the expression of Runx1 was detected by RT-PCR and Western blot respectively. The recombinant adenovirus Ad-Runx1 overexpressing Runx1 was constructed and verified at the mRNA and protein levels Ad-Runx1. The MEFs were treated with Ad-Runx1 and BMP9 conditioned medium to detect alkaline phosphatase (ALP) staining and activity in the early stage of osteogenesis. Alizarin red S staining was used to detect calcium deposition in the late stage of osteogenesis. RT- The mRNA and protein levels of Runx2, a key osteoblast transcription factor, were detected by PCR and Western blot respectively. RESULTS: Runx1 mRNA and protein levels were up-regulated in MEFs treated with Ad-BMP9. Runx1 mRNA and protein levels were up-regulated in Ad-Runx1-treated MEFs. BMP-induced MEFs were treated with Ad-Runx1 , Enhanced ALP activity and calcium deposition as well as Runx2 mRNA and protein expression. Conclusion: Runx1 can promote BMP9-induced mesenchymal stem cell MEFs osteogenic differentiation.