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  

Pulse Electromagnetic Field Promotes Rat Osteoblast Maturation and Mineralization via Activation of the NO/cGMP Pathway

作　　者：任茜[1] 王鸣刚[1] 陈克明[2] 周建[2] 石文贵[2] 方清清 REN Qian WANG Ming-Gang CHEN Ke-Ming ZHOU Jian SHI Wen-Gui FANG Qing-Qing(） College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China ） Institute of Orthopaedics, Lanzhou General Hospital of PLA, Lanzhou 730050, China)

机构地区：[1]兰州理工大学生命科学与工程学院,兰州730050 [2]兰州军区兰州总医院骨科研究所,兰州730050

出　　处：《中国生物化学与分子生物学报》2016年第11期1242-1248,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81270963);甘肃省自然科学基金(No.1506RJZA306)资助~~

摘　　要：脉冲电磁场(pulse electromagnetic fields,PEMFs)能够促进大鼠成骨细胞(rat osteoblast cells,ROB)成熟矿化,但是其作用机制并不明确。本实验主要研究PEMFs促进大鼠成骨细胞成熟矿化与NO/c GMP信号途径的关系,进而阐明PEMFs促进成骨细胞成熟矿化的机理。首先,将成骨细胞经50 Hz、0.6 m T脉冲电磁场作用不同时间后,检测细胞培养液中一氧化氮(nitric oxide,NO)和细胞内3'-5'-环鸟苷一磷酸(3'-5'-cyclic-GMP,c GMP)的含量,以探明电磁场是否影响NO和c GMP的合成;其次,应用蛋白质印迹,检测细胞内e NOS、i NOS和PKG-1的蛋白表达量;最后,利用NOS的阻断剂L-NAME抑制NO信号通路后,检测成骨性相关指标,包括碱性磷酸酶(ALP)活性、钙化结节数量、成骨性基因Bmp-2、Collagen-1、Osterix及破骨细胞调节因子Rankl基因的表达量。结果发现,经PEMFs处理后,NO含量及c GMP含量均有明显升高;细胞内e NOS、i NOS和PKG-1蛋白表达量较空白对照组均有显著升高,说明PEMFs能够激活NO/c GMP信号途径。且经PEMFs处理的成骨细胞,ALP活性升高,BMP-2、Collagen-1和Osterix基因表达量显著增加,Rankl基因表达量下降,成骨细胞形成钙化结节的能力增强。当加入L-NAME,PEMFs引起的ALP活性增加、成骨性基因表达升高和钙化结节形成能力增强的趋势均被显著抑制。上述结果表明,经PEMFs处理成骨细胞成熟矿化过程中,NO/c GMP信号通路被激活;如该通路被抑制,则电磁场促成骨作用被抵消,说明脉冲电磁场促进成骨细胞成熟矿化依赖于NO/c GMP信号通路。Pulse electromagnetic fields （PEMFs） have been considered as a stimulator for rat osteoblasts （ROB） maturation and mineralization. However, the mechanism of the PEMFs-induced osteogenesis is still unknown. In this study, we examined the role of the NO/cGMP signaling pathway in PEMFs-induced maturation and mineralization of primary ROB. First, the ROB was exposed at the PEMFs of 50 Hz 0.6 mT in different time, then NO was detected in cell culture and the intracellular cGMP content, to find out whether the PEMFs effects the NO and cGMP synthesis. Second, we collected total protein, detected the eNOS,iNOS and PKG-1 protein expression by Western blot. Finally, we used the L-NAME to block the NO signaling pathway, and then detected the bone-related indicators. We also detected ALP activity and gene expression of Bmp-2, Collagen-1,0sterix and Rankl. The results showed that by PEMFs treatment, the NO and cGMP concentration was raised; and the eNOS, iNOS, PKG-1 protein expression was also improved. With the effect of PEMFs, ALP activity was increased; Bmp-2, collagen-1 and osterrix gene expression was increased, the gene of Rankl was decreased, the formation of calcified nodule increased capacity. When with addition of L-NAME inhibitor, all the phenomena disappeared. Collectively, we demonstrated that after exposing at the PEMFs, the NO/cGMP pathway was activated. If the pathway was inhibited, the osteogeuesis of PEMFs was offset. The results showed that PEMFs promoted osteoblast maturation and mineralization by activated the NO/cGMP signaling pathway.

关 键 词：脉冲电磁场 大鼠成骨细胞 成熟与矿化 一氧化氮 环鸟苷一磷酸 

分 类 号：R580[医药卫生—内分泌] R138[医药卫生—内科学]