北京大学学报(医学版) ›› 2019, Vol. 51 ›› Issue (2): 245-251. doi: 10.19723/j.issn.1671-167X.2019.02.008

• 论著 • 上一篇    下一篇

脉冲电磁场促进成骨细胞成熟分化依赖于初级纤毛-PI3K/AKT途径

任茜1,周建1,王鸣刚2,陈克明1,()   

  1. 1. 兰州军区兰州总医院骨科研究所,兰州 730050
    2. 兰州理工大学生命科学与工程学院,兰州 730050
  • 收稿日期:2017-05-17 出版日期:2019-04-18 发布日期:2019-04-26
  • 通讯作者: 陈克明 E-mail:chkeming@yahoo.com.cn
  • 基金资助:
    国家自然科学基金(81270963);国家自然科学基金(81471090);甘肃省自然科学基金(1506RJZA306)

Pulsed electromagnetic fields stimulating osteogenic differentiation and maturation involves primary cilia-PI3K/AKT pathway

Qian REN1,Jian ZHOU1,Ming-gang WANG2,Ke-ming CHEN1,()   

  1. 1. Institute of Orthopaedics, Lanzhou General Hospital of PLA, Lanzhou 730050, China
    2. College of Life Science and Engineering,Lanzhou University of Technology, Lanzhou 730050, China
  • Received:2017-05-17 Online:2019-04-18 Published:2019-04-26
  • Contact: Ke-ming CHEN E-mail:chkeming@yahoo.com.cn
  • Supported by:
    the National Natural Science Foundation of China(81270963);the National Natural Science Foundation of China(81471090);Gansu Natural Science Foundation(1506RJZA306)

摘要:

目的: 研究脉冲电磁场(pulse electromagnetic fields, PEMF)促进大鼠成骨细胞成熟分化是否与初级纤毛和PI3K/AKT途径相关,探讨PEMF促进骨形成的作用机制。方法: 用酶解法获取新生SD大鼠颅骨成骨细胞(rat calvarial osteoblasts, ROB), 50 Hz、0.6 mT PEMF处理0、0.5、1、1.5和2 h后检测磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K)和蛋白激酶B(protein kinase B, AKT)蛋白表达量及初级纤毛长度和发生率的变化情况;用LY294002阻断PI3K/AKT信号途径,观察PEMF促进ROB成骨性分化是否受到影响;以RNAi法干扰IFT88的基因表达以抑制初级纤毛发生,观察PEMF激活的PI3K/AKT信号途径及ROB的成骨性分化是否受到影响。成骨性分化指标包括碱性磷酸酶(alkaline phosphatase, ALP)活性,real-time PCR法和Western blot法检测的成骨性相关基因BMP-2、COL-1和OSX的基因表达量,以及钙化结节数量等。结果: 经PEMF处理0、0.5、1、1.5、2 h后,ROB的PI3K、AKT蛋白表达量升高(P<0.01),初级纤毛变长;其中PI3K在0.5 h时蛋白表达量达到最高,随着PEMF处理时间增加,蛋白表达量降低; AKT在0.5 h和1.5 h时蛋白表达量较高。用PI3K阻断剂LY294002阻断PI3K/AKT信号途径后,PEMF不再能提高成骨细胞中ALP 活性和成骨性相关基因BMP-2、COL-1、OSX的基因表达量,但在阻断前PEMF能增加ROB中ALP活性和成骨性相关基因的表达;RNAi干扰初级纤毛发生后,PEMF不再能增加PI3K的蛋白表达量,说明初级纤毛干扰后,PEMF不能激活PI3K/AKT信号途径;其次,PEMF提高ALP活性的作用消失,也不能提高BMP-2、COL-1和OSX的基因表达量,其增加ROB钙化结节形成能力的作用也消失,说明初级纤毛干扰后,PEMF促进成骨细胞成熟矿化的能力消失。结论: PEMF通过成骨细胞表面的初级纤毛激活了PI3K/AKT信号途径,进而发挥了骨形成活性的促进作用。

关键词: 脉冲电磁场, 成骨细胞, RNA干扰, PI3K

Abstract:

Objective: To study whether the pulsed electromagnetic fields (PEMF) promoting rat osteoblasts differentiation and maturation is related to the primary cilia and PI3K/AKT pathway, and to explore the mechanism of PEMF in promoting bone differentiation.Methods: Enzyme solution was used to obtain newborn SD rats calvarial osteoblasts (ROB), which were processed by 50 Hz 0.6 mT PEMF for 0, 0.5, 1, 1.5 and 2 h, detecting PI3K and AKT protein expression and changes in primary cilia length and incidence; with LY294002 blocking PI3K/AKT signaling pathways we observed whether PEMF promoted osteogenic differentiation of ROB was affected; by interfering IFT88 gene expression by RNAi to inhibit primary cilia we observed whether PI3K/AKT signaling pathway and osteogenic differentiation of ROB was affected. Osteogenic differentiation indexes included alkaline phosphatase (ALP) activity, Real-time PCR and Western blot detection of osteogenic related genes of BMP-2, COL-1 and OSX and calcified nodules number, etc.Results: After exposure to PEMF for 0, 0.5, 1, 1.5, and 2 h, the protein expression of PI3K and AKT in ROB were increased significantly (P<0.01) and the primary cilia became longer; and the protein expression of PI3K reached the highest level at 0.5 h, as the treatment time of PEMF increased, the PI3K protein expression decreased. AKT showed higher protein expression at 0.5 h and 1.5 h. After blocking the PI3K/AKT signaling pathway with the PI3K blocker LY294002, PEMF could no longer increase ALP activity and the gene expressions of BMP-2, COL-1, OSX which were osteogenically related. However, PEMF could increase the ALP activity and the osteogenically related gene expression in ROB before blocking. After RNAi interfered the primary cilia, PEMF could no longer increase the protein expression of PI3K, which indicated that PEMF could not activate the PI3K/AKT signaling pathway after primary cilia interfering; secondly, the effect of PEMF on enhancing ALP activity disappeared, it also decrease the gene expressions of BMP-2, COL-1, and OSX, and the ability of increasing the calcification nodule formation also disappeared, indicating that the ability of PEMF to promote osteoblast maturation and mineralization disappeared after primary cilia interference.Conclusion: PEMF activated the PI3K/AKT signaling pathway through primary cilia on the surface of osteoblasts, then promoted bone formation activity and differentiation.

Key words: Pulse electromagnetic fields (PEMF), Osteoblast, RNA interference, PI3K

中图分类号: 

  • R336

图1

电磁场对初级纤毛的影响"

图2

50Hz 0.6mT 电磁场能够增强PI3K蛋白表达量"

图3

50 Hz 0.6 mT 电磁场增强p-AKT蛋白表达量"

图4

电磁场和LY294002对大鼠成骨细胞ALP活性的影响"

图5

电磁场和LY294002对大鼠成骨细胞成骨性基因表达的影响"

"

图7

干扰IFT88基因后对大鼠成骨细胞初级纤毛的影响"

图8

电磁场和RNAi对PI3K蛋白表达的影响"

图9

电磁场和RNAi对ALP活性的影响"

图10

电磁场和RNAi对成骨性相关基因的影响"

图11

IFT88 siRNA介导的初级纤毛抑制对PEMF诱导大鼠颅骨成骨细胞矿化成熟的影响"

[1] 石文贵, 马小妮, 陈克明 . 初级纤毛在细胞信号转导中的作用与机制[J]. 浙江大学学报(医学版), 2014,43(3):359-365.
doi: 10.3785/j.issn.10089292.2014.04.006
[2] Shi WG, Xie YF, He JP , et al. Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia[J]. Sci Rep, 2017,7(1):1866-1877.
doi: 10.1038/s41598-017-02049-9
[3] Wanka H, Lutze P, Staar D , et al.( Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia[J]. J Cell Mol Med, 2017,21(7):13-19.
doi: 10.1111/jcmm.2017.21.issue-1
[4] Zhai YK, Guo XY, Ge BF , et al. Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K-AKT-eNOS-NO-cGMP-PKG[J]. Bone, 2014,66(9):189-198.
doi: 10.1016/j.bone.2014.06.016
[5] Ehnert S, Sreekumar V, Asperawerz RH , et al. TGF-β1 impairs mechanosensation of human osteoblasts via HDAC6-mediated shortening and distortion of primary cilia[J]. J Mol Med, 2017,95(6):1-11.
[6] Kirschen GW, Liu H, Lang T , et al. The radial organization of neuronal primary cilia is acutely disrupted by seizure and ischemic brain injury[J]. Front Biol, 2017,12(2):1-15.
doi: 10.1007/s11515-016-1438-7
[7] Akama KT , McEwen BS. Estrogen stimulates postsynaptic density-95 rapid protein synjournal via the Akt /protein kinase B pathway[J]. J Neurosci, 2003,23(6):2333-2339.
doi: 10.1523/JNEUROSCI.23-06-02333.2003
[8] Markus A, Zhong J, Snider WD . Raf and akt mediate distinct aspects of sensory axon growth[J]. Neuron, 2002,35(1):65-76.
doi: 10.1016/S0896-6273(02)00752-3
[9] 孟金兰, 陈雅嘉, 陈红 , 等. PI3K/Akt 信号通路在硫化氢保护PC12细胞对抗化学性缺氧损伤的作用[J]. 中国药理学通报, 2013,29(2):257-261.
[10] Chen Y, Hu Y, Yang L , et al. Runx2 alleviates high glucose-suppressed osteogenic differentiation VIA PI3K/AKT/GSK3β/β-catenin pathway[J]. Cell Biol Int, 2017,41(8):822-832.
doi: 10.1002/cbin.v41.8
[11] Ma Y, Fu S, Lu L , et al. Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways[J]. Mol Cell Endocrinol, 2017,45(8):83-93.
[12] Wang J, Ma XY, Feng YF , et al. Magnesium lons promote the biological behaviour of rat calvarial osteoblasts by activating the PI3K/Akt signalling pathway[J]. Biol Trace Elem Res, 2017,179(2):1-10.
doi: 10.1007/s12011-017-0938-x
[13] Song F, Wang Y, Jiang D , et al. Cyclic compressive stress regulates apoptosis in rat osteoblasts: involvement of PI3K/Akt and JNK MAPK signaling pathways[J]. PLoS One, 2016,11(11):165-176.
[14] Zhou J, Ming LG, Ge BF , et al. Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation differentiation and mineralization potentials of rat osteoblasts[J]. Bone, 2011,49(4):753-761
doi: 10.1016/j.bone.2011.06.026
[15] 高玉海, 成魁, 葛宝丰 , 等. 50Hz不同强度正弦交变电磁场对大鼠峰值骨量的影响[J]. 生物医学工程学杂志, 2015,13(1):116-119
[16] 任茜, 王鸣刚, 陈克明 , 等. 脉冲电磁场促进大鼠成骨细胞成熟与矿化依赖于NO/cGMP信号途径[J]. 中国生物化学与分子生物学报, 2016,32(11):1242-1248.
[17] 方清清, 李志忠, 陈克明 , 等. 50Hz 1.8mT正弦交变电磁场通过调节成骨细胞PGE2分泌影响Opg/Rankl的基因表达[J]. 中国生物化学与分子生物学报, 2015,18(9):983-988.
[18] Liu JM, Ning G, Chen JL . Osteoporotic fractures in Asia: risk factors and strategies for prevention[J]. J Bone Miner Metab, 2007,25(1):1-5.
[19] 张辉, 李亮, 王茜 , 等. 骨形成蛋白-7对多孔钽/软骨细胞复合物分泌功能以及 Col-Ⅱ、AGG 和 Sox9基因表达的影响[J]. 北京大学学报(医学版), 2015,47(2):219-225.
doi: 10.3969/j.issn.1671-167X.2015.02.006
[20] Paula FJ, Rosen CJ . Back to the future: revisiting parathyroid hormone and calcitonin control of bone remodeling[J]. Horm Metab Res, 2010,42(5):299-306.
doi: 10.1055/s-0030-1248255
[21] Bonnelye E, Chabadel A, Saltel F , et al. Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro[J]. Bone, 2008,42(1):129-138.
doi: 10.1016/j.bone.2007.08.043
[22] 何伟, 李自力, 崔元璐 , 等. 淫羊藿苷对大鼠成骨细胞核结合因子α1、骨形成蛋白-2、骨形成蛋白-4 mRNA表达的影响[J]. 北京大学学报(医学版), 2009,41(6):669-673.
[23] Bassett CA . The development and application of pulsed electromagnetic fields (PEMFs) for ununited fractures and arthrodeses[J]. Orthop Clin North Am, 1984,15(1):61-87.
[24] Spasic M, Jacobs CR . Lengthening primary cilia enhances cellular mechanosensitivity[J]. Eur Cells Master, 2017,33(2):158-169.
doi: 10.22203/eCM
[25] Yan JL, Zhou J, Ma HP , et al. Pulsed electromagnetic fields promote osteoblast mineralization and maturation needing the existence of primary cilia[J]. Mol Cell Endocrinol, 2015,404(3):132-140
doi: 10.1016/j.mce.2015.01.031
[26] Xie YF, Shi WG, Zhou J , et al. Pulsed electromagnetic fields stimulate osteogenic differentiation and maturation of osteoblasts by upregulating the expression of BMPRⅡ localized at the base of primary cilium[J]. Bone, 2016,93(2):22-32.
doi: 10.1016/j.bone.2016.09.008
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