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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (04) : 197 -203. doi: 10.3877/cma.j.issn.1674-1366.2017.04.002

所属专题: 文献

基础研究

NELL-1调控RUNX2的P1启动子诱导成骨分化
张弘1, 姚宇1, 孙佳栋1, 于潇楠1, 张志光1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
  • 收稿日期:2016-06-23 出版日期:2017-08-01
  • 通信作者: 张志光
  • 基金资助:
    国家自然科学基金(81400491); 广东省医学科研基金(A2014254); 中山大学高校青年教师培育项目(15ykpy41)

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

Hong Zhang1, Yu Yao1, Jiadong Sun1, Xiaonan Yu1, Zhiguang Zhang1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2016-06-23 Published:2017-08-01
  • Corresponding author: Zhiguang Zhang
  • About author:
    Corresponding author: Zhang Zhiguang, Email:
引用本文:

张弘, 姚宇, 孙佳栋, 于潇楠, 张志光. NELL-1调控RUNX2的P1启动子诱导成骨分化[J/OL]. 中华口腔医学研究杂志(电子版), 2017, 11(04): 197-203.

Hong Zhang, Yu Yao, Jiadong Sun, Xiaonan Yu, Zhiguang Zhang. NELL-1 regulates the P1 promoter of RUNX2 to promote osteogenic differentiation[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(04): 197-203.

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(FN800= 26.979,PN800<0.05;FN1600= 28.410,PN1600<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(FN800= 31.179,PN800<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 factor NELL-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 were determined by EGFP gene expression analysis using flow cytometry. Alizarin Red Staining Quantification Assay 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-time PCR. Data were analyzed using One-Way ANOVA procedure.

Results

The 800 ng/ml or 1600 ng/ml NELL-1-induced increase of P1 promoter of RUNX2 by 82.09% or 92.36% was statistically significant, compared with control (FN800= 26.979, PN800<0.05; FN1600= 28.410, PN1600<0.01) . Alizarin Red Staining Quantification Assay indicated that 800 ng/ml NELL-1 group in calcium compounds was dramatically reduced by 73.41%, compared with control (FN800= 31.179, PN800<0.01) ; Real-time PCR detected that NELL-1 can promote the expression of OPN, OCN, ALP and RUNX2 during the differentiation process in mouse mesenchymal stem cells.

Conclusion

NELL-1 enhance the osteoblastic differentiation of mouse MSCs by promote the expression of RUNX2 by its P1 promoter.

图1 RUNX2的P1启动子EGFP慢病毒载体示意图
表1 实时定量PCR各引物序列
图2 RUNX2的P1启动子报告基因绿色荧光蛋白表达情况
图3 不同浓度NELL-1蛋白组和BMP-2蛋白组表达绿色荧光细胞数量情况
图4 茜素红染色检测NELL-1蛋白诱导M2-10B4细胞成骨实验
图5 实时定量PCR检测NELL-1对小鼠M2-10B4细胞成骨相关基因表达的影响
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