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Title: 持続的な機械的刺激はRAW264.7細胞における破骨細胞分化を抑制する
Other Titles: Continuous mechanical stress suppresses osteoclastogenesis in RAW264.7 cells
Authors: 上村, 光太郎1 Browse this author
吉村, 善隆2 Browse this author →KAKEN DB
南川, 元3 Browse this author
鈴木, 邦明4 Browse this author →KAKEN DB
飯田, 順一郎5 Browse this author →KAKEN DB
Authors(alt): Uemura, Kotaro1
Yoshimura, Yoshitaka2
Minamikawa, Hajime3
Suzuki, Kuniaki4
Iida, Junichiro5
Keywords: 破骨細胞
mechanical stress
nitric oxide (NO)
Issue Date: Mar-2018
Publisher: 北海道歯学会
Journal Title: 北海道歯学雑誌
Volume: 38
Issue: 2
Start Page: 169
End Page: 176
Abstract: 骨の恒常性を保つために機械的刺激が重要な役割を果たしていることが明らかにされつつあるが,破骨細胞の分化誘導系に対し機械的刺激を直接作用させた報告は我々の研究のみである.これまで我々は周期的な機械的刺激による破骨細胞融合の抑制に関して報告してきた.本研究では持続的な機械的刺激が破骨細胞分化誘導系に与える影響について検討した.RANKL添加培養液を用いてRAW264.7細胞をBioFlex plateにて通法に従い培養した.Flexcell tension systemを用いて破骨細胞数の増加が顕著に見られる培養4日目から2日間,伸展率5,10,15 %で刺激を与えると,10,15%の伸展率で破骨細胞数の有意な減少が認められた.また,1,3,6,12 h/日,2日間,伸展率10 %で持続的な機械的刺激を作用させた結果,6,12 hにおいて破骨細胞数の有意な減少が認められた.持続的な機械的刺激は,周期的な機械的刺激と同様に破骨細胞分化誘導系の抑制が認められた.TRAP陽性破骨細胞を2-4核,5核以上で分類し計測した結果,機械的刺激により2-4核の破骨細胞数では経時的な減少が認められた.また,実験群と対照群のNO量を測定した結果,実験群でNO量の有意な増加が認められた.2-4核の破骨細胞数の減少にNOが関与することが示唆されたため,NO合成酵素阻害剤(L-NMMA)で処理した結果,2-4核の破骨細胞数の減少が抑制され,破骨細胞数の減少にNOが関与することが示唆された.また,realtime-PCR法によりmRNAの発現量を測定した結果,破骨細胞の融合因子の1つであるDC-STAMPの有意な減少が認められたが,iNOS,nNOSに有意な差は認められなかった.以上の結果から持続的な機械的刺激は破骨細胞の融合を抑制させる要因の一つであり,その一部にNOが関与していることが示唆された.
Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Our previous studies have been among the few in which the direct effects of mechanical stress on osteoclastogenesis were assessed. Our reports have shown inhibition of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effect of continuous mechanical stress on osteoclastogenesis. We cultured RAW264.7 cells stimulated with the receptor activator of nuclear factor-κB ligand (RANKL) using a BioFlex plate. Osteoclasts were stimulated using a Flexcell tension system at elongation rates of 5 %, 10 %, and 15 % for two days. (This was 4 days after the culture was initiated.) On the second day a notable increase in the number of osteoclasts was found. The number of osteoclasts showed significant decreases compared with control osteoclasts after stimulation at elongation rates of 10 % and 15 %. Osteoclasts were also stimulated at an elongation rate of 10 % for 1, 3, 6, and 12 h each day for two days, and significant decreases in the numbers of osteoclasts were found for osteoclasts that were stimulated for 6 h and 12 h. Continuous mechanical stress inhibited osteoclastogenesis were found for cyclic mechanical stress. We counted the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts that had 2-4 nuclei and 5 or more nuclei. We found that the number of osteoclasts with 2-4 nuclei was decreased by continuous mechanical stress. Measurement of the quantity of NO showed that the quantity increased in stimulated osteoclasts compared to that in control osteoclasts. Since the results suggested that NO was related to the decrease in the number of osteoclasts with 2-4 nuclei, we treated the cells with an NOS inhibitor (L-NMMA). Treatment with L-NMMA suppressed the decrease in number of osteoclasts with 2-4 nuclei. Measurement of mRNA expression by real-time PCR showed that the quantity of dendritic cell-specific transmembrane protein (DC-STAMP), which is one of the fusion factors for osteoclasts, was significantly decreased in stimulated osteoclasts compared with that in the control osteoclasts. However, there were no changes in inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). The results suggest that continuous mechanical stress is one of the factors that inhibit fusion of osteoclasts and that NO is involved in the inhibition.
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