Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (2): 158-164    DOI: 10.3785/j.issn.1008-9292.2019.04.06
原著     
低频脉冲电磁场通过IGF-1R/NO信号通路促进大鼠颅骨成骨细胞成熟及矿化
邵佳乐1,2(),李志忠2,周建1,李凯1,秦荣1,陈克明1,*()
1. 中国人民解放军联勤保障部队第九四〇医院骨科研究所, 甘肃 兰州 730050
2. 兰州理工大学生命科学与工程学院, 甘肃 兰州 730050
Effect of low-frequency pulsed electromagnetic fields on activity of rat calvarial osteoblasts through IGF-1R/NO signaling pathway
SHAO Jiale1,2(),LI Zhizhong2,ZHOU Jian1,LI Kai1,QIN Rong1,CHEN Keming1,*()
1. Institute of Orthopedic Research, the 940th Hospital of Joint Logistics Support Force of People's Liberation Army, Lanzhou 730050, China
2. School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
 全文: PDF(1008 KB)   HTML( 6 )
摘要：

目的: 观察低频脉冲电磁场（PEMF）是否通过胰岛素样生长因子1受体（IGF-1R）/一氧化氮（NO）信号通路促进大鼠颅骨成骨细胞成熟及矿化。方法: 体外分离培养乳鼠颅骨成骨细胞，传代后随机分成9组，每天用50 Hz 0.6 mT PEMF处理不同的时间，通过检测成骨细胞中NO含量确定PEMF最佳处理时长。将传代后的成骨细胞随机分成空白对照组、PEMF组、GSK（IGF-1R阻断剂）组、PEMF+GSK组，PEMF处理成骨细胞3 d后，采用蛋白质印迹法检测蛋白激酶（AKT）、诱导型一氧化氮合酶（iNOS）和cGMP依赖性蛋白激酶（PKG）蛋白表达水平；PEMF处理第6天时测定碱性磷酸酶（ALP）活性；PEMF处理第12天时采用茜红素染色法观察钙化结节形成情况。结果: 经1.5 h电磁场处理后的成骨细胞中NO含量显著提高（P < 0.01），遂后续采用PEMF处理1.5 h。与空白对照组比较，PEMF组AKT、iNOS、PKG蛋白表达量增加（均P < 0.01），ALP活性升高（P < 0.05），且茜红素染色面积增大（P < 0.01）；GSK组AKT、iNOS、PKG蛋白表达量减少，ALP活性降低（P < 0.05），茜红素染色面积最小（P < 0.01）；PEMF+GSK组上述实验结果均高于GSK组但低于PEMF组（P < 0.05或P < 0.01）。结论: PEMF可能通过IGF-1R/NO信号通路促进大鼠颅骨成骨细胞成熟与矿化。
关键词： 成骨细胞/辐射效应;  颅骨/细胞学;  电磁场;  糖原合成酶激酶3/生物合成;  受体, IGF1型/生物合成;  信号传导;  细胞, 培养的;  大鼠, Wistar    
Abstract:

Objective: To investigate the effect of low-frequency pulsed electromagnetic fields (PEMF) on the maturation and mineralization of rat cranial osteoblasts in vitro and its relation to IGF-1R/NO signaling pathway. Methods: The rat osteoblasts were isolated and cultured in vitro and randomly divided into blank control group, PEMF group, GSK group (IGF-1R blocker) and PEMF+GSK group. The cells were treated with 50 Hz 0.6 mT PEMF for 1.5 h/d. After 3 d of PEMF treatment, the expressions of protein kinase (AKT), inducible nitric oxide synthase (iNOS) and cGMP-dependent protein kinase (PKG) were detected by Western blotting; on 6 d of PEMF treatment alkaline phosphatase (ALP) activity was determined; on 12 d of PEMF treatment the calcification nodule formation was demonstrated by Alizarin red staining. Results: NO level was significantly increased in rat osteoblasts treated with 50 Hz 0.6 mT PEMF for 1.5 h/d. Western blot analysis showed that the expressions of AKT, iNOS and PKG protein in PEMF group were higher than those in the control group (all P < 0.01); the ALP activity was increased(P < 0.05), and the PEMF group had the largest area of Alizarin red staining (P < 0.01). The expressions of AKT, iNOS and PKG protein in GSK group were lower than those in the control group; the ALP activity was decreased (P < 0.05), and the GSK group had the least area of Alizarin red staining (P < 0.01). The expressions of AKT, iNOS, PKG protein, the ALP activity and the area of Alizarin red staining in PEMF+GSK group were between PEMF group and GSK group. Conclusion: PEMF may enhance the maturation and mineralization of rat cranial osteoblasts in vitro through IGF-1R/NO signaling pathway.
Key words: Osteoblasts/radiation effects    Skull/cytology    Electromagnetic fields    Glycogen synthase kinase 3/biosynthesis    Receptor, IGF type 1/biosynthesis    Signal transduction    Cells, cultured    Rats, Wistar
收稿日期: 2018-10-15 出版日期: 2019-07-24
CLC:  Q683  
基金资助: 国家自然科学基金(81770879);国家国际科技合作专项(2015DFR3094)
通讯作者: 陈克明     E-mail: shaojiale1119@163.com;chenkm@lut.cn
作者简介: 邵佳乐(1993-), 女, 硕士研究生, 主要从事电磁场对骨代谢影响研究; E-mail:shaojiale1119@163.com; https://orcid.org/0000-0002-2399-0110
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
邵佳乐
李志忠
周建
李凯
秦荣
陈克明

引用本文:

邵佳乐,李志忠,周建,李凯,秦荣,陈克明. 低频脉冲电磁场通过IGF-1R/NO信号通路促进大鼠颅骨成骨细胞成熟及矿化[J]. 浙江大学学报（医学版）, 2019, 48(2): 158-164.

SHAO Jiale,LI Zhizhong,ZHOU Jian,LI Kai,QIN Rong,CHEN Keming. Effect of low-frequency pulsed electromagnetic fields on activity of rat calvarial osteoblasts through IGF-1R/NO signaling pathway. J Zhejiang Univ (Med Sci), 2019, 48(2): 158-164.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.04.06    或    http://www.zjujournals.com/med/CN/Y2019/V48/I2/158

图 1  不同时间低频脉冲电磁场处理的大鼠原代颅骨成骨细胞一氧化氮含量变化
图 2  低频脉冲电磁场处理的成骨细胞AKT、iNOS和PKG蛋白表达量变化
图 3  低频脉冲电磁场干预第12天时各组钙化结节茜红素染色面积
1 马俊岭, 郭海英, 阳晓东 . 骨质疏松症的流行病学概况[J]. 中国骨质疏松杂志, 2009, 38 (18): 1744- 1746
MA Junling , GUO Haiying , YANG Xiaodong . Epidemiological overview of osteoporosis[J]. Chinese Journal of Osteoporosis, 2009, 38 (18): 1744- 1746
2 HOROWITE M C . Cytokines and estrogen in bone:anti-osteoporotic effects[J]. Science, 1993, 260 (5108): 626- 627
doi: 10.1126/science.8480174
3 宋涛, 霍小林, 吴石增 . 生物电磁特性及其应用[M]. 北京: 北京工业大学出版社, 2008: 1- 12
SONG Tao , HUO Xiaolin , WU Shizeng . Electromagnetic properties and its application[M]. Beijing: Beijing University of Technology Press, 2008: 1- 12
4 孙文均, 姜槐 . 电磁场生物效应的细胞膜及蛋白激酶信号转导机制研究[J]. 国际生物医学工程杂志, 2004, 27 (5): 260- 263
SUN Wenjun , JIANG Huai . Effects of electromagnetic field on biological membrane and protein kinase signal transduction mechanism[J]. Biomedical Engineering Foreign Medical Sciences, 2004, 27 (5): 260- 263
doi: 10.3760/cma.j.issn.1673-4181.2004.05.002
5 KULAR J , TICKNER J , CHIM S M et al. An overview of the regulation of bone remodelling at the cellular level[J]. Clin Biochem, 2012, 45 (12): 863- 873
doi: 10.1016/j.clinbiochem.2012.03.021
6 DEL S C , GHIOE S , LUSCHI P et al. Pain perception and electromagnetic fields[J]. Neurosci Biobehav Rev, 2007, 31 (4): 619- 642
doi: 10.1016/j.neubiorev.2007.01.003
7 赵敏, 许建中, 周强 et al. 脉冲电磁场诱导人骨髓间充质干细胞向成骨细胞分化的研究[J]. 中华物理医学与康复杂志, 2005, 27 (1): 5- 8
ZHAO Min , XU Jianzhong , ZHOU Qiang et al. Pulsed electromagnetic field induces differentiation of human bone marrow mesenchymal stem cells into osteoblasts[J]. Chinese Journal of Physical Medicine and Rehabilitation, 2005, 27 (1): 5- 8
doi: 10.3760/j:issn:0254-1424.2005.01.003
8 TSAI M T , CHANG W H , CHANG K et al. Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering[J]. Bioelectromagnetics, 2007, 28 (7): 519- 528
doi: 10.1002/(ISSN)1521-186X
9 ZHOU J , CHEN K , BAOFENG G E et al. Double effect intensity of 50 Hz sinusoidal electromagnetic fields at different intensity on differentiation and mRNA expression of osteoblasts in vitro[J]. Acta Biophysica Sinica, 2011, 27 (6): 507- 516
10 杨海燕, 侯建明, 吴曼 et al. 乳铁蛋白对大鼠成骨细胞增殖及IGF-1/IGF-1R mRNA表达的影响[J]. 中华骨质疏松和骨矿盐疾病杂志, 2014, 7 (1): 35- 41
YANG Haiyan , HOU Jianming , WU Man et al. Effect of lactoferrin on proliferation of rat osteoblasts and expression of IGF-1/IGF-1R mRNA[J]. Chinese Journal of Osteoporosis and Bone Mineral Research, 2014, 7 (1): 35- 41
doi: 10.3969/j.issn.1674-2591.2014.01.007
11 任茜, 周建, 石文贵 et al. 环磷酸鸟苷及其相关信号通路在骨代谢过程中的作用[J]. 解放军医药杂志, 2016, 28 (10): 20- 22
REN Qian , ZHOU Jian , SHI Wengui et al. Role of cyclic guanosine monophosphate and its related signaling pathways in bone metabolism[J]. Medical & Pharmaceutical Journal of Chinese People's Liberation Army, 2016, 28 (10): 20- 22
12 CHENG G , ZHAI Y , CHEN K et al. Sinusoidal electromagnetic field stimulates rat osteoblast differentiation and maturation via activation of NO-cGMP-PKG pathway[J]. Nitric Oxide, 2011, 25 (3): 316- 325
doi: 10.1016/j.niox.2011.05.009
13 ZHAI Y K , GUO X Y , GE B F 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
14 周建, 葛宝丰, 陈克明 et al. 正弦交变电磁场促进体外培养成骨细胞成熟矿化的时间效应[J]. 生物医学工程学杂志, 2011, 28 (6): 1085- 1088
ZHOU Jian , GE Baofeng , CHEN Keming et al. Time effect of sinusoidal electromagnetic field on enhancing the maturation and mineralization of osteoblasts in vitro[J]. Journal of Biomedical Engineering, 2011, 28 (6): 1085- 1088
15 MEDIERO A , CRONSTEIN B N . Adenosine and bone metabolism[J]. Trends Endocrinol Metab, 2013, 24 (6): 290- 300
doi: 10.1016/j.tem.2013.02.001
16 MARKOV M S . "Biological Windows":a tribute to W. Ross Adey[J]. Environmentalist, 2005, 25 (2-4): 67- 74
doi: 10.1007/s10669-005-4268-8
17 ATALAY Y , GUNES N , GUNER M D et al. Pentoxifylline and electromagnetic field improved bone fracture healing in rats[J]. Drug Des Devel Ther, 2015, 9:5195- 5201
18 BASSETT C A , PILLA A A , PAWLUK R J . A non-operative salvage of surgically-resistant pseudarthroses and non-unions by pulsing electromagnetic fields. A preliminary report[J]. Clin Orthop Relat Res, 1977, (124): 128- 143
19 何成奇, 王维, 肖登 et al. 不同治疗时间脉冲电磁场对去势大鼠血清雌二醇及股骨骨钙含量的影响[J]. 中国康复医学杂志, 2007, 22 (4): 1078- 1080
HE Chengqi , WANG Wei , XIAO Deng et al. Effects of pulsed electromagnetic fields at different treatment times on serum estradiol and femur bone calcium in ovariectomized rats[J]. China Journal of Rehabilitation Medicine, 2007, 22 (4): 1078- 1080
20 高玉海, 成魁, 葛宝丰 et al. 50 Hz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, 32 (1): 116- 119, 136
GAO Yuhai , CHENG Kui , GE Baofeng et al. Effect of 50Hz different intensity sinusoidal electromagnetic field on peak bone mass in rats[J]. Journal of Biomedical Engineering, 2015, 32 (1): 116- 119, 136
21 LUBEN R A , CAIN C D , CHEN M C et al. Effects of electromagnetic stimuli on bone and bone cells in vitro:inhibition of responses to parathyroid hormone by low-energy low-frequency fields[J]. Proc Natl Acad Sci U S A, 1982, 79 (13): 4180- 4184
doi: 10.1073/pnas.79.13.4180
22 BLANK M , GOODMAN R . Do electromagnetic fields interact directly with DNA?[J]. Bioelectromagnetics, 1997, 18 (2): 111- 115
doi: 10.1002/(ISSN)1521-186X
23 BRIGHTON C T , MCCLUSKEY W P . Response of cultured bone cells to a capacitively coupled electric field:inhibition of cAMP response to parathyroid hormone[J]. J Orthop Res, 1988, 6 (4): 567- 571
doi: 10.1002/(ISSN)1554-527X
24 AI S , CHENG X W , INOUE A et al. Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase[J]. Biochem Biophys Res Commun, 2007, 364 (2): 395- 401
doi: 10.1016/j.bbrc.2007.10.027
25 L?SCHINGER M , THUMM S , H?MMERLE H et al. Induction of intracellular calcium oscillations in human skin fibroblast populations by sinusoidal extremely low-frequency magnetic fields (20 Hz, 8 mT) is dependent on the differentiation state of the single cell[J]. Radiat Res, 1999, 151 (2): 195- 200
doi: 10.2307/3579770
26 LOHMANN C H , SCHWARTZ Z , LIU Y et al. Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells[J]. J Orthop Res, 2003, 21 (2): 326- 334
doi: 10.1016/S0736-0266(02)00137-7
27 CHANG W H , CHEN L T , SUN J S et al. Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities[J]. Bioelectromagnetics, 2004, 25 (6): 457- 465
doi: 10.1002/(ISSN)1521-186X
28 JING D , ZHAI M , TONG S et al. Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in bone defect repair through a Wnt/β-catenin signaling-associated mechanism[J]. Sci Rep, 2016, 6:32045
doi: 10.1038/srep32045
29 XIE Y F , SHI W G , 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, 93:22- 32
doi: 10.1016/j.bone.2016.09.008
30 FITZSIMMONS R J , RYABY J T , MAGEE F P et al. IGF-Ⅱ receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields[J]. J Bone Miner Res, 1995, 10 (5): 812- 819
31 ZHU D , SHI S , WANG H et al. Growth arrest induces primary-cilium formation and sensitizes IGF-1-receptor signaling during differentiation induction of 3T3-L1 preadipocytes[J]. J Cell Sci, 2009, 122 (Pt 15): 2760- 2768
32 任茜, 王鸣刚, 陈克明 et al. 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
REN Qian , WANG Minggang , CHEN Keming et al. Pulse electromagnetic field promotes rat osteoblast maturation and mineralization via activation of the NO/cGMP pathway[J]. Chinese Journal of Biochemistry Molecular Biology, 2016, 32 (11): 1242- 1248
33 BAUD'HUIN M , LAMOUREUX F , DUPLOMB L et al. RANKL, RANK, osteoprotegerin:key partners of osteoimmunology and vascular diseases[J]. Cell Mol Life Sci, 2007, 64 (18): 2334- 2350
doi: 10.1007/s00018-007-7104-0
34 闫娟丽, 王鸣刚, 陈克明 et al. 不同强度50Hz脉冲电磁场促进大鼠颅骨成骨细胞矿化成熟最佳参数的筛选[J]. 中国生物化学与分子生物学报, 2014, 30 (7): 721- 729
YAN Juanli , WANG Minggang , CHEN Keming et al. Screening 50Hz pulaed electromagnetic fields of different intensities on proliferation, differentiation and mineralization of rat osteoblast for the optimal parameters[J]. Chinese Journal of Biochemistry Molecular Biology, 2014, 30 (7): 721- 729
[1] 王青梅, 徐千姿, 魏安怡, 陈世硕, 张翀, 曾玲晖. 大剂量维生素C通过减少糖酵解和蛋白质合成抑制乳腺癌细胞增殖[J]. 浙江大学学报（医学版）, 2019, 48(3): 296-302.
[2] 朱紫菱, 谈静, 邓红. 肿瘤细胞膜/质蛋白转位入核研究进展[J]. 浙江大学学报（医学版）, 2019, 48(3): 318-325.
[3] 梁刚, 牛育苗, 李一涵, 魏安怡, 董静尹, 曾玲晖. 雷帕霉素在大鼠局灶性脑缺血再灌注后24 h给药对脑损伤的保护作用[J]. 浙江大学学报（医学版）, 2018, 47(5): 443-449.
[4] 朱锋,范苗,徐孜惟,蔡依廷,陈益臻,余双,曾玲晖. 雷帕霉素对帕金森病小鼠的保护作用[J]. 浙江大学学报（医学版）, 2018, 47(5): 465-472.
[5] 钱波,张彦玲,莫绪明. 先天性食管闭锁相关转录因子及信号通路研究进展[J]. 浙江大学学报（医学版）, 2018, 47(3): 239-243.
[6] 潘宗富,方琦璐,张轶雯,李莉,黄萍. 基于生物信息学的未分化甲状腺癌关键发病机制及其潜在干预靶点研究[J]. 浙江大学学报（医学版）, 2018, 47(2): 187-193.
[7] 丁京京,卢韵碧. 受体相互作用蛋白家族在炎症中的作用研究进展[J]. 浙江大学学报（医学版）, 2018, 47(1): 89-96.
[8] 刘婷婷,王凌霄,杨晓辉,姚智卿,蔡慧珍. MyD88非依赖性信号通路在枸杞多糖抑制糖尿病小鼠肿瘤坏死因子α中的作用[J]. 浙江大学学报（医学版）, 2018, 47(1): 35-40.
[9] 朱保应,周建,高玉海,石文贵,魏振龙,李文苑,王媛媛,陈克明. 低频率低强度电磁场对体外培养大鼠颅骨成骨细胞成熟和矿化的影响[J]. 浙江大学学报（医学版）, 2017, 46(6): 585-592.
[10] 魏振龙,石文贵,陈克明,周建,王鸣刚. 淫羊藿素通过CXCR4/SDF-1信号通路促进小鼠成骨细胞成熟和矿化[J]. 浙江大学学报（医学版）, 2017, 46(6): 571-577.
[11] 张斌斌 等. 抑制哺乳动物雷帕霉素靶蛋白信号通路对慢性脑缺血小鼠认知功能的改善和机制[J]. 浙江大学学报（医学版）, 2017, 46(4): 405-412.
[12] 周延峰 等. 1.8 mT不同频率正弦电磁场对青年大鼠骨生物力学性能的影响[J]. 浙江大学学报（医学版）, 2016, 45(6): 561-567.
[13] 方清清 等. 低频脉冲电磁场促进成骨细胞分化的基因调节和非基因调节探究[J]. 浙江大学学报（医学版）, 2016, 45(6): 568-574.
[14] 封盛 等. 糖皮质激素受体信号通路在膀胱癌治疗中的作用研究进展[J]. 浙江大学学报（医学版）, 2016, 45(6): 655-660.
[15] 崔光莹等. 脉冲电场肿瘤消融激发免疫反应的研究进展[J]. 浙江大学学报（医学版）, 2015, 44(6): 672-677.