Journal of Prevention and Treatment for Stomatological Diseases ›› 2020, Vol. 28 ›› Issue (1): 16-23.DOI: 10.12016/j.issn.2096-1456.2020.01.003

• Basic Study • Previous Articles     Next Articles

The regulatory mechanisms of IGF1 in the osteogenic differentiation of canine MSMSCs via BMP2-Smad1/5 signaling pathway

LIAO Chunhui1,LI Mingfei2,YE Jinmei1,PENG Wei2,CHEN Songling2()   

  1. 1.Department of Orthodontics, Guangzhou Women and Children′s Medical Center, Guangzhou 510000, China
    2.Department of Stomatology, The First Affiliated Hospital, SunYat-sen University, Guangzhou 510080, China
  • Received:2019-07-17 Revised:2019-09-23 Online:2020-01-20 Published:2020-01-17
  • Contact: Songling CHEN

IGF1通过BMP2-Smad1/5信号通路调控犬上颌窦黏膜干细胞成骨分化

廖春晖1,李明飞2,叶金梅1,彭伟2,陈松龄2()   

  1. 1.广州市妇女儿童医疗中心口腔正畸科,广东 广州(510000)
    2.中山大学附属第一医院口腔科,广东 广州(510080)
  • 通讯作者: 陈松龄
  • 作者简介:廖春晖,主治医师,硕士,Email:954222137@qq.com
  • 基金资助:
    广东省自然科学基金项目(2018A030313173);国家自然科学基金项目(81371111);广州市妇女儿童医疗中心儿科研究所内部科研基金项目(YIP-2018-017)

Abstract:

Objective To investigate the role of the bone morphogenetic protein 2 (BMP2)-Smad1/5 and p38MAPK signaling pathways in the osteogenic differentiation of MSMSCs by insulin-like growth factor 1 (IGF1).Methods A recombinant adenovirus (RAD) and IGF1 expressing IGF1 gene were constructed. After osteogenic induction, qRT-PCR and Western blot were used to detect the phosphorylation level of Smad1/5 and the expression of the BMP-2 protein in the BMP-Smad signaling pathway; immunohistochemistry was used to observe the nuclear translocation of Smad1/5; qRT-PCR and Western blot were used to detect IGF with Noggin and SB203580, inhibitors of the p38MAPK signaling pathway 1-mediated osteogenic differentiation of MSMSCs.Results The recombinant IGF1 adenovirus was constructed successfully. MSMSCs were cultured in inductive medium after infection with different concentrations of Ad-IGF1, and then, the protein levels of BMP2 and p-Smad1/5 increased. IGF1 can also induce nuclear translocation of Smad1/5. In addition, Noggin significantly reduced the phosphorylation level of Smad1/5 and the formation of mineralized nodules in the MSMSCs. The mRNA levels of Runx2, OPN and ALP also decreased. In contrast, SB203580 decreased neither the phosphorylation level of p38 nor the mRNA expression of Runx2, OPN and ALP in the Ad-IGF1 MSMSCs.Conclusion IGF1 can promote the osteogenic differentiation of MSMSCs via the BMP2-Smad1/5 signaling pathway. In contrast, IGF1 may not promote the osteogenic differentiation of MSMSCs via the p38MAPK signaling pathway.

Key words: insulin-like growth factor 1, maxillary sinus membrane stem cells, BMP2-Smad1/5 signaling pathway, p38 MAPK signaling pathway, osteogenic differentiation, Runx2, osteopontin, alkaline phosphatase

摘要:

目的 探讨骨形态形成蛋白2(bone morphogenetic protein2,BMP2)-Smad1/5及p38MAPK信号通路在胰岛素样生长因子1(insulin-like growth factor 1,IGF1)介导的促犬上颌窦黏膜干细胞(maxillary sinus membrane stem cells,MSMSCs)成骨分化中的作用。方法 构建表达胰岛素样生长因子1(insulin-like growth factor1,IGF1)基因的重组腺病毒载体(recombinant adenovirus,rAdv)Ad-IGF1。感染Ad-IGF1的犬上颌窦黏膜干细胞,经成骨诱导培养后,qRT-PCR和Western blot检测BMP-Smads信号通路中重要信号蛋白Smad1/5的磷酸化水平和BMP2蛋白的表达;免疫组化观察磷酸化Smad1/5核转位情况;qRT-PCR及Western blot 检测BMP-Smads通路抑制剂Noggin和p38MAPK信号通路抑制剂SB203580对IGF1介导的促犬MSMSCs成骨分化的影响。结果 成功构建IGF1基因表达重组腺病毒载体Ad-IGF1;感染Ad-IGF1的犬MSMSCs,经成骨诱导培养后,Smad1/5的磷酸化水平和BMP2蛋白的表达升高,IGF1可促使Smad1/5核转位;BMP-Smads信号通路抑制剂Noggin可抑制Smad1/5的磷酸化,降低成骨标志物Runx2、OPN和ALP mRNA的表达,钙结节形成减少。p38MAPK信号通路抑制剂SB203580不能降低Ad-IGF1 犬MSMSCs的p38磷酸化水平,亦不能降低成骨标志物Runx2、OPN和ALP mRNA的表达。结论 犬MSMSCs成骨分化过程中,IGF1通过经典的Smads 蛋白依赖性信号转导通路BMP2-Smad1/5促进成骨,而Smads蛋白非依赖性信号转导通路p38MAPK在IGF1介导的犬MSMSCs成骨过程中可能并不发挥作用。

关键词: 胰岛素样生长因子1, 上颌窦黏膜干细胞, BMP2-Smad1/5信号通路, p38MAPK信号通路, 成骨分化, Runx2, 骨桥蛋白, 碱性磷酸酶

CLC Number: