NELL-1调控RUNX2的P1启动子诱导成骨分化

NELL-1 regulates the P1 promoter of RUNX2 to promote osteogenic differentiation

目的探讨骨生长因子Nel样蛋白1(NELL-1)与骨核心转录因子RUNX2之间的调控机制。方法构建并产生含RUNX2的P1启动子和绿色荧光蛋白报告基因的慢病毒,感染小鼠骨髓间充质细胞系M2-10B4细胞,通过流氏细胞仪检测绿色荧光蛋白细胞记数值,观察不同浓度外源NELL-1蛋白对RUNX2的P1启动子的调控作用。茜素红染色半定量法检测NELL-1促进M2-10B4细胞成骨作用以及实时定量聚合酶链反应(PCR)检测NELL-1蛋白对小鼠M2-10B4细胞成骨相关基因表达的影响。采用单因素方差分析数据。结果当加入800和1600 ng/ml NELL-1蛋白时,NELL-1蛋白组绿色荧光蛋白细胞记数值分别较对照组增高82.09%、92.36%,且差异均有统计学意义(F_(N800)=26.979,P_(N800)<0.05;F_(N1600)=28.410,P_(N1600)<0.01)。茜素红染色定量检测结果表明,800 ng/ml NELL-1组小鼠M2-10B4细胞较对照组产生的矿化结节升高约73.41%,差异具有统计学意义(F_(N800)=31.179,P_(N800)<0.01)。实时定量PCR结果显示,NELL-1蛋白促进成骨相关基因骨核心转录因子RUNX2、骨桥蛋白(OPN)、骨钙蛋白(OCN)和碱性磷酸酶(ALP)表达上调。结论 NELL-1蛋白可以通过调控RUNX2的P1启动子促进RUNX2表达诱导骨髓间充质细胞成骨分化。

Objective To investigate regulation mechanism between the osteoinductive factorNELL-1 and the osteogenic transcription factor RUNX2. Methods The EGFP lentivirus vector with the P1 promoter of RUNX2 were constructed,obtained and transfected the M2-10B4 cells. The activity of the P1 Promoter of RUNX2 after the treatment of different doses of BMP-2 protein or NELL-1 protein weredetermined by EGFP gene expression analysis using flow cytometry. Alizarin Red Staining QuantificationAssay evaluated the calcium nodules in three groups during the osteoblastic differentiation of mouse MSCs.The bone formation-related genes OPN,OCN,ALP and RUNX2 expression were detected by real-timePCR. Data were analyzed using One-Way ANOVA procedure. Results The 800 ng/ml or 1600 ng/mlNELL-1-induced increase of P1 promoter of RUNX2 by 82.09% or 92.36% was statistically significant,compared with control（F_（N800）= 26.979,P_（N800）〈0.05;F_（N1600）= 28.410,P_（N1600）〈0.01）. Alizarin Red StainingQuantification Assay indicated that 800 ng/ml NELL-1 group in calcium compounds was dramaticallyreduced by 73.41%,compared with control（F_（N800）= 31.179,P_（N800）〈0.01）;Real-time PCR detected thatNELL-1 can promote the expression of OPN,OCN,ALP and RUNX2 during the differentiation process inmouse mesenchymal stem cells. Conclusion NELL-1 enhance the osteoblastic differentiation of mouseMSCs by promote the expression of RUNX2 by its P1 promoter.