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浙江大学学报（医学版）
浙江大学学报（医学版）  2017, Vol. 46 Issue (6): 585-592    DOI: 10.3785/j.issn.1008-9292.2017.12.03
骨组织代谢及再生专题     
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
朱保应(),周建,高玉海,石文贵,魏振龙,李文苑,王媛媛,陈克明*()
兰州军区兰州总医院全军骨科中心骨科研究所, 甘肃 兰州 730050
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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摘要：

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    
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 words: Osteoblasts/cytology    Skull/cytology    Electromagnetic fields    Cell differentiation
收稿日期: 2017-09-21 出版日期: 2017-12-25
CLC:  Q683  
基金资助: 国家自然科学基金(81270963, 81471090);甘肃省自然科学基金(1506RJZA306);甘肃省空间辐射生物学重点实验室开放基金(2016GKL002)
通讯作者: 陈克明     E-mail: zby19930118@126.com;chenkm@lut.cn
作者简介: 朱保应(1993-), 男, 硕士研究生, 主要从事低频电磁场防治骨质疏松症的研究; E-mail:zby19930118@126.com; https://orcid.org/0000-0002-3232-120X
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朱保应
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引用本文:

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.

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.

链接本文:

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

图 1  实验所用电磁场发生装置及电磁场波形图
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
表 1  实时定量RT-PCR引物序列
图 2  电磁场干预2 d时各组Collagen-1、BMP-2、OSX和Runx-2蛋白电泳图
图 3  电磁场干预2 d时各组Collagen-1、BMP-2、OSX和Runx-2蛋白表达量比较
图 4  电磁场干预2 d时各组Collagen-1、BMP-2、OSX和Runx-2基因表达量比较
图 5  电磁场干预后不同时间各组碱性磷酸酶(ALP)活性变化
图 6  电磁场干预第10天各组ALP偶氮偶合染色结果
图 7  电磁场干预第12天时各组钙化结节茜素红染色结果
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