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J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (6): 585-592    DOI: 10.3785/j.issn.1008-9292.2017.12.03
    
Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts in vitro
ZHU Baoying(),ZHOU Jian,GAO Yuhai,SHI Wengui,WEI Zhenlong,LI Wenyuan,WANG Yuanyuan,CHEN Keming*()
Institute of Orthopedic Research, Lanzhou General Hospital of PLA, Lanzhou 730050, China
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Abstract  

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P < 0.01 or P < 0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P < 0.05), while such difference was not observed in SEMF group (P>0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P < 0.05), but the difference between PEMF and SEMF groups was not significant (P>0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P < 0.01), and the stained area was bigger in PEMF group than that in SEMF group (P < 0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P < 0.01), and more calcium nodules were observed in PEMF group than SEMF group (P < 0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.



Key wordsOsteoblasts/cytology      Skull/cytology      Electromagnetic fields      Cell differentiation     
Received: 21 September 2017      Published: 25 December 2017
CLC:  Q683  
  R68  
Corresponding Authors: CHEN Keming     E-mail: zby19930118@126.com;chenkm@lut.cn
Cite this article:

ZHU Baoying,ZHOU Jian,GAO Yuhai,SHI Wengui,WEI Zhenlong,LI Wenyuan,WANG Yuanyuan,CHEN Keming. Effect of low frequency low intensity electromagnetic fields on maturation and mineralization of rat skull osteoblasts in vitro. J Zhejiang Univ (Med Sci), 2017, 46(6): 585-592.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2017.12.03     OR     http://www.zjujournals.com/med/Y2017/V46/I6/585


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

目的: 比较研究50 Hz 1.8 mT正弦电磁场(SEMF)和50 Hz 0.6 mT脉冲电磁场(PEMF)对大鼠颅骨成骨细胞成熟和矿化的影响。方法: 原代培养大鼠颅骨成骨细胞,传代后随机分为空白对照组、SEMF组和PEMF组。SEMF组和PEMF组每天接受电磁场处理一次,每次90 min。电磁场处理2 d时,采用蛋白质印迹法和实时定量RT-PCR检测Ⅰ型胶原(Collagen-1)、骨形态发生蛋白2(BMP-2)、Osterix(OSX)和Runt相关转录因子2(Runx-2)的蛋白和基因表达;电磁场处理第6天和第9天时,采用碱性磷酸酶(ALP)试剂盒检测ALP活性;电磁场处理第10天时采用偶氮偶合染色检测ALP活性;电磁场处理第12天时,以茜素红染色观察钙化结节形成情况并作定量分析。结果: 与空白对照组比较,SEMF组和PEMF组的Collagen-1、BMP-2、OSX、Runx-2蛋白和基因表达水平均显著升高(P < 0.01或P < 0.05),且Collagen-1、BMP-2在PEMF组中的mRNA表达量显著高于SEMF组(均P < 0.05)。电磁场处理第6天时,PEMF组ALP活性显著高于空白对照组(P < 0.05),SEMF组与空白对照组比较差异无统计学意义(P>0.05);第9天时,PEMF组和SEMF组ALP活性均显著高于空白对照组(均P < 0.01),而PEMF组和SEMF组之间差异无统计学意义(P>0.05)。PEMF组和SEMF组偶氮偶合染色面积均大于空白对照组(均P < 0.01),且PEMF组染色面积大于SEMF组(P < 0.05)。茜素红染色结果显示,PEMF组和SEMF组钙化结节染色面积显著大于空白对照组(均P < 0.01),且PEMF组钙化结节染色面积大于SEMF组(P < 0.01)。结论: 50 Hz 1.8 mT SEMF和50 Hz 0.6 mT PEMF均能促进大鼠颅骨成骨细胞成熟和矿化,其中PEMF效果更为明显。


关键词: 成骨细胞/细胞学,  颅骨/细胞学,  电磁场,  细胞分化 
Fig 1 Magnetic field generator and electromagnetic oscillographs
基因 引物序列(5′→3′) 产物长度(bp)
Gapdh 正向:GGCACAGTCAAGGCTGAGAATG
反向:ATGGTGGTGAAGACGCCAGTA
143
Bmp-2 正向:GATGTCACCCGGCTTGTGAT
反向:TCGTCGTGGCCAAAACTCA
191
Collagen-1 正向:TGGCAAGGCCGGTGAAGATA
反向:TGACCAGGAAGCCCTCGGACT
249
Osx 正向:GCCTACTTACCCGTCTGACTTT
反向:GCCCACTATTGCCAACTGC
131
Runx-2 正向:GCACCCAGCCCATAATAGA
反向:TTGGAGCAAGGAGAACCC
165
Tab 1 Primer sequences for real-time RT-PCR
Fig 2 Electropherogram of Collagen-1, BMP-2, OSX and Runx-2 expression in each group
Fig 3 Protein expressions of Collagen-1, BMP-2, OSX and Runx-2 in each group
Fig 4 mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in each group
Fig 5 Activities of alkaline phosphatase (ALP) in rat calvarial osteoblasts at different time after electromagnetic field treatment
Fig 6 Immunohistochemical results of ALP in rat calvarial osteoblasts after 10 days of electromagnetic field treatment
Fig 7 Alizarin red staining showed calcium nodule in rat calvarial osteoblasts after 12 days of electromagnetic field treatment
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