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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (02) : 65 -72. doi: 10.3877/cma.j.issn.1674-1366.2017.02.001

所属专题: 文献

基础研究

大鼠骨髓间充质干细胞成牙本质向分化中Wnt信号通路磷酸化抗体芯片分析
黄艳玲1, 郑健茂1, 凌均棨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:2017-02-08 出版日期:2017-04-01
  • 通信作者: 凌均棨

Analysis on Wnt signaling phospho antibody array in the process of rat bone marrow mesenchymal stem cells differentiated into odontoblast-like cells

Yanling Huang1, Jianmao Zheng1, Junqi Ling1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2017-02-08 Published:2017-04-01
  • Corresponding author: Junqi Ling
  • About author:
    Corresponding author: Ling Junqi, Email:
引用本文:

黄艳玲, 郑健茂, 凌均棨. 大鼠骨髓间充质干细胞成牙本质向分化中Wnt信号通路磷酸化抗体芯片分析[J/OL]. 中华口腔医学研究杂志(电子版), 2017, 11(02): 65-72.

Yanling Huang, Jianmao Zheng, Junqi Ling. Analysis on Wnt signaling phospho antibody array in the process of rat bone marrow mesenchymal stem cells differentiated into odontoblast-like cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(02): 65-72.

目的

研究大鼠骨髓间充质干细胞(BMSC)成牙本质向分化过程中,Wnt信号通路主要信号分子的磷酸化变化,初步探讨其作用机制。

方法

全骨髓贴壁法培养大鼠BMSC,利用新生大鼠牙胚制备牙胚细胞条件培养基,诱导大鼠BMSC向成牙本质向分化,应用Wnt信号通路磷酸化抗体芯片技术检测诱导组与对照组Wnt信号通路蛋白磷酸化水平并筛选出调变磷酸化位点,应用蛋白印迹法(Western blot)验证芯片结果。采用SPSS 20.0软件进行统计分析,采用独立样本t检验和校正t检验分析组间差异。

结果

全骨髓贴壁法获得自我更新和多向分化潜能的BMSC,经牙胚细胞条件培养基诱导2 d后,细胞形态开始转变为梭形,7 d后形成长梭形并螺旋状排布生长,成牙本质相关蛋白DSPP(t= 3.700,P= 0.021)、DMP1(t= 3.237,P= 0.032)、RUNX2(t= 12.09,P<0.001)、ALP(t= 62.83,P<0.001)表达水平上调,成功诱导BMSC向成牙本质向分化,Wnt信号通路磷酸化抗体芯片检测发现27个磷酸化调变位点,Western blot检测结果显示核内β-catenin表达上调(t= 4.588,P= 0.044),PKCδ磷酸化水平上调(t= 5.438,P= 0.032)、AKT磷酸化水平下调(t= 30.85,P= 0.001),与芯片结果一致。

结论

通过Wnt信号通路磷酸化抗体芯片分析,获得BMSC成牙本质向分化过程中,Wnt信号通路磷酸化位点变化。

Objective

To investigate the changes of protein phosphorylation level of Wnt signaling in the process of rat bone marrow mesenchymal stem cells (BMSCs) differentiated into odontoblast-like cell and preliminarily discuss the mechanism.

Methods

We seperated, cultured and purified BMSCs by the method of whole bone marrow adherence. Tooth germ cell-conditioned medium was used to induce the differentiation of rats bone marrow mesenchymal stem cells into odontoblast-like cells. A phospho-specific antibody microarray (PNT227) targeting the Wnt Phospho signaling pathway was used to find the alterations of the downstream signaling events. Western blot was carried out to validate the antibody array results. The SPSS 20.0 software was used as the data processing tool. T-test and t'-test were used to analyze the difference between the groups.

Results

BMSCs obtained by whole bone marrow adherence method had the potential of self-renew and multilineage differentiation. After co-culture with TGC-CM for 2 days, BMSCs began to exhibit long spindle process. After 7 days, the cells had a long spindle shape and a tightly packed arrangement. The expression of DSPP (t = 3.700, P= 0.021) , DMP1 (t = 3.237, P= 0.032) , RUNX2 (t = 12.09, P<0.001) , ALP (t = 62.83, P<0.001) were enhanced after induction for 7 days. Results of Wnt signaling phospho antibody array showed 27 phosphorylation sites were up-regulated or down-regulated. Western blot analysis showed that nuclear expression of β-catenin (t= 4.588, P= 0.044) and the phosphorylation level of PKCδ (t= 5.438, P= 0.032) were enhanced, while the phosphorylation level of AKT (t= 30.85, P= 0.001) was reduced, which was consistent with the array.

Conclusion

Changes of phosphorylation site of Wnt signaling in the process of BMSCs differentiated into odontoblast-like cells were found.

图1 倒置显微镜观察骨髓间充质干细胞形态特点(× 50)
图2 P3代骨髓间充质干细胞生长曲线
图3 骨髓间充质干细胞成骨及成脂分化
图4 骨髓间充质干细胞经成牙本质向诱导后的形态变化(× 50)
图5 Western blot检测骨髓间充质干细胞经成牙本质向诱导后DSPP、DMP1、RUNX2、ALP蛋白表达变化
表1 Wnt磷酸化抗体芯片中调变的27个磷酸化位点及比值
图6 Wnt信号通路磷酸化抗体芯片扫描图像
图7 Western blot检测骨髓间充质干细胞经成牙本质向诱导后胞核β-catenin蛋白表达变化以及PKCδ和AKT蛋白磷酸化水平变化
[1]
Shahdadfar A, Fronsdal K, Haug T,et al. In vitro expansion of human mesenchymal stem cells:choice of serum is a determinant of cell proliferation,differentiation,gene expression,and transcriptome stability[J]. Stem Cells,2005,23(9):1357-1366.
[2]
Ye L, Chen L, Feng F,et al. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells[J]. Cell Biol Int,2015,39(10):1151-1161.
[3]
Liu F, Chu EY, Watt B,et al. Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis[J]. Dev Biol,2008,313(1):210-224.
[4]
Zhang YM, Dai BL, Zheng L,et al. A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation[J]. Cell Death Dis,2012(3):e406.
[5]
Pittenger MF, Mackay AM, Beck SC,et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science,1999,284(5411):143-147.
[6]
Krause DS, Theise ND, Collector MI,et al. Multi-organ,multi-lineage engraftment by a single bone marrow-derived stem cell [J]. Cell,2001,105(3):369-377.
[7]
Long X, Olszewski M, Huang W,et al. Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells[J]. Stem Cells Dev,2005,14(1):65-69.
[8]
Makino S, Fukuda K, Miyoshi S,et al. Cardiomyocytes can be generated from marrow stromal cells in vitro[J]. J Clin Invest,1999,103(5):697-705.
[9]
Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis:embryonic events as a template for dental tissue repair?[J]. Crit Rev Oral Biol Med,2001,12(5):425-437.
[10]
Wang J, Liu B, Gu S,et al. Effects of Wnt/β-catenin signalling on proliferation and differentiation of apical papilla stem cells[J]. Cell Prolif,2012,45(2):121-131.
[11]
Scheller EL, Chang J, Wang CY. Wnt/beta-catenin inhibits dental pulp stem cell differentiation[J]. J Dent Res,2008,87(2):126-130.
[12]
Shan T, Zhou C, Yang R,et al. Lithium chloride promotes the odontoblast differentiation of hair follicle neural crest cells by activating Wnt/β-catenin signaling[J]. Cell Biol Int,2015,39(1):35-43.
[13]
Etheridge SL, Spencer GJ, Heath DJ,et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells[J]. Stem Cells,2004,22(5):849-860.
[14]
Gao Q, Xia Y, Liu L,et al. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity[J]. Sci Rep,2016(6):26577.
[15]
Sheng L, Mao X, Yu Q,et al. Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells[J]. Exp Ther Med,2017,13(1):55-62.
[16]
He H, Zhao ZH, Han FS,et al. Activation of protein kinase C ε enhanced movement ability and paracrine function of rat bone marrow mesenchymal stem cells partly at least independent of SDF-1/CXCR4 axis and PI3K/AKT pathway[J]. Int J Clin Exp Med,2015,8(1):188-202.
[17]
Lee S, Cho HY, Bui HT,et al. The osteogenic or adipogenic lineage commitment of human mesenchymal stem cells is determined by protein kinase C delta[J]. BMC Cell Biol,2014(15):42.
[18]
Wang Y, Yi XD, Li CD. Suppression of mTOR signaling pathway promotes bone marrow mesenchymal stem cells differentiation into osteoblast in degenerative scoliosis:in vivo and in vitro[J]. Mol Biol Rep,2017,44(1):129-137.
[19]
Zhang LL, Liu JJ, Liu F,et al. MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway[J]. Biochem Biophys Res Commun,2012,420(4):875-881.
[20]
董金山,段晴月,文军,等.骨髓间充质干细胞向成牙本质样细胞分化的体外实验研究[J].牙体牙髓牙周病学杂志,2010,20(4):192-195.
[21]
Yu Y, Wang L, Yu J,et al. Dentin matrix proteins(DMPs)enhance differentiation of BMMSCs via ERK and P38 MAPK pathways[J]. Cell Tissue Res,2014,356(1):171-182.
[22]
Thornton TM, Pedraza-Alva G, Deng B,et al. Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation[J]. Science,2008,320(5876):667-670.
[23]
Chang J, Sonoyama W, Wang Z,et al. Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK[J]. J Biol Chem,2007,282(42):30938-30948.
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