口腔疾病防治 ›› 2020, Vol. 28 ›› Issue (4): 219-223.DOI: 10.12016/j.issn.2096-1456.2020.04.003

• 基础研究 • 上一篇    下一篇

mTORC1信号通路在张应力下对小鼠骨髓间充质细胞成骨分化的作用

彭海艳,蒋校文(),黄华庆,陈金勇   

  1. 南方医科大学附属郴州市第一人民院口腔科 南华大学转化医学研究所,湖南 郴州(423000)
  • 收稿日期:2019-06-24 修回日期:2019-08-25 出版日期:2020-04-20 发布日期:2020-03-31
  • 通讯作者: 蒋校文
  • 作者简介:彭海艳,硕士,副主任医师,Email:359227119@qq.com
  • 基金资助:
    国家自然基金(81301651);湖南省自然科学基金(2018JJ2015);郴州市第一人民医院重点项目(N2019-003)

The role of the mTORC1 signaling pathway during osteogenic differentiation of mouse bone marrow mesenchymal cells under tension stress

PENG Haiyan,JIANG Xiaowen(),HUANG Huaqing,CHEN Jinyong   

  1. Department of Stomatology, The First People′s Hospital of Chenzhou City, The South Medical University & Institute of Translation Medicine, University of China South, Chenzhou 423000, China
  • Received:2019-06-24 Revised:2019-08-25 Online:2020-04-20 Published:2020-03-31
  • Contact: Xiaowen JIANG

摘要:

目的 探讨哺乳动物雷帕霉素复合物1(mammalian target of rapamycin complex 1,mTORC1)信号通路在周期性单轴牵张力作用下对小鼠骨髓间充质细胞(bone marrow mesenchymal cells,BMMSCs)的成骨分化的作用。方法 对体外分离培养的小鼠BMMSCs施加形变量10%的单轴动态牵张力,在牵张后0、1、2、4、8 h利用western blot检测内源性mTORC1信号通路主要分子mTOR、Raptor、核糖体蛋白S6激酶(ribosomal proteinS6 kinases,S6K)的表达变化,并利用化学比色法检测碱性磷酸酶(alkaline phosphatase,ALP)的活性变化,ELISA法检测检测骨钙素(osteocalcin,OCN)表达量,RT-PCR法检测Runt相关转录因子(Runt-related transcription factor 2,Runx2)的mRNA表达变化。将BMMSCs分为抑制组、激活组及对照组,分别加入工具药PP242、MHY1485及PBS,加力2 h后用上述方法检测S6K与成骨信号相关因子的活性或表达变化。结果 Western blot结果显示,mTORC1信号通路主要分子在牵张力作用后的8 h内均有表达,并在加力后2 h表达最高。与对照组相比,抑制mTORC1信号通路表达后,ALP活性、OCN表达下降,Runx2 的mRNA水平上升,差异均具有统计学意义(P<0.001);激活mTORC1信号通路表达后,ALP活性、OCN表达上升,而Runx2 mRNA水平下降,差异均具有统计学意义(P<0.001)。结论 mTORC1信号通路参与了牵张力下小鼠BMMSCs成骨分化过程,激活该信号通路可以促进其成骨分化。

关键词: 骨髓间充质细胞, 哺乳动物雷帕霉素靶蛋白复合体1, 周期性单轴牵张力, 牵张成骨, 碱性磷酸酶, 骨钙素, Runt相关转录因子, 核糖体蛋白S6激酶

Abstract:

Objective To investigate the expression of the mTORC1 signaling pathway during the osteogenic differentiation of mouse bone marrow mesenchymal cells (BMMSCs) under cyclic uniaxial tension and explore its possible role. Methods The BMMSCs of mice were affected by uniaxial dynamic tensile force. Western blot was used to detect the expression changes of major molecules (mTOR, Raptor, S6K) in the endogenous mTORC1 signaling pathway at 0, 1, 2, 4, and 8 hours after stretching. Chemical colorimetry, ELISA and PCR were used to detect alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2 mRNA, respectively. Then, inhibition, activation and control groups were established by administration of the drugs PP242, MHY1485 and PBS, respectively. Two hours after the stress, the expression of S6K was detected by western blot, and the expression of the osteogenic signal was continuously detected by the above methods. Results Western blot analysis showed that the main molecules of the mTORC1 signaling pathway were all expressed within 8 hours after traction, and the highest expression was 2 hours after the stress. Compared with those in the control group, the ALP activity and OCN expression decreased and the Runx2 mRNA levels increased after the mTORC1 signal pathway was inhibited (P < 0.001); ALP activity and OCN expression increased after the mTORC1 signal pathway was activated, while the Runx2 mRNA levels decreased (P < 0.001). Conclusion The mTORC1 signaling pathway participates in the osteogenic differentiation of mouse BMMSCs under tension. The osteogenesis of BMMSCs under cyclic uniaxial tension would be enhanced if the mTORC1 signaling pathway was activated.

Key words: bone marrow mesenchymal cells, mammalian rapamycin target protein complex 1, cyclic uniaxial tension, distraction osteogenesis, alkaline phosphatase, osteocalcin, Runt related transcription factors, ribosomal protein S6 kinase

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