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間歇的な機械的刺激は RAW264.7 細胞において破骨細胞分化を抑制する
加藤, 結香;吉村, 善隆;上村, 光太郎;南川, 元;鈴木, 邦明;飯田, 順一郎
Permalink : http://hdl.handle.net/2115/75689
KEYWORDS : 間歇的機械的刺激;破骨細胞;CD47;intermittent mechanical stress;osteoclast
Abstract
骨改造において機械的刺激が重要な役割を果たしていることが明らかとなっているが，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および持続的な機械的刺激による破骨細胞分化の抑制に関して報告してきた．本研究では，可撤式矯正装置の間歇的な使用による組織変化を想定して，間歇的な機械的刺激を加えた場合の破骨細胞分化への影響について検討した．RAW 264.7 細胞を通法に従い３日間培養した．その後，Flexcell tension system を用い，培養４日目から48時間，伸展率10 %で，合計刺激時間を24時間とし，1，2，3，4，6，12時間毎にそれぞれ持続的機械的刺激と無刺激とを繰り返し，間歇的な機械的刺激とした．48時間無刺激としたものを対照群とした．TRAP 染色にて核数ごとに破骨細胞数を測定し，破骨細胞関連遺伝子の mRNA 発現量をリアルタイム PCR 法にて定量した．全ての実験群では破骨細胞数が有意に抑制された．また，１時間毎の刺激付与群（１時間群）と12時間毎の刺激付与群（12時間群）を比較すると，1時間群において総破骨細胞数は有意に抑制された．特に2，3，4核の破骨細胞数が有意に抑制された．両群のmRNA 発現量の比較では，DC-STAMP および OC-STAMP，CD47 の発現は１時間群において有意に抑制された．12時間群に比べ１時間群では，単核の前破骨細胞同士，および単核の前破骨細胞と２～４核の小数核の破骨細胞との融合に関与している CD47 の発現抑制により，２～４核の破骨細胞数が抑制され，さらに DC-STAMP および OC-STAMP の発現抑制により総破骨細胞数が抑制されることが示唆された．以上から，間歇的な機械的刺激を付与する回数，時間によって細胞融合因子が抑制され，破骨細胞分化を抑制することが示唆された．
Mechanical stress plays an important role in the remodeling of bone. In previous reports by this research team, we showed the suppression of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effects of intermittent 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 10 % for 2 days from the fourth day of culture. In different treatments, mechanical stress was applied for 24 h, followed by bouts of continuous mechanical stress and no stress alternating at 1, 2, 3, 4, 6, or 12 h. In addition, we assayed osteoclast-related genetic mRNA expression using the real-time PCR method. We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts with 2, 3, 4, 5, 6, or 7 nuclei, and those with more nuclei. The number of osteoclasts was lower after mechanical stress when compared with no mechanical stress. The number of total osteoclasts was significantly suppressed in the group stimulated every hour (1-h group) when compared with the group that was stimulated every 12 h (12-h group). Furthermore, we found that the number of osteoclasts with 2-4 nuclei was significantly reduced by intermittent continuous mechanical stress. The measurement of mRNA expression using real-time PCR showed the suppression of DC-STAMP, OC-STAMP, and CD47, which are fusion factors for osteoclasts. These significantly decreased in the 1-h group compared with that in 12-h group. CD47 participates in cell fusion with the mononuclear preosteoclasts among each other, and among the mononuclear preosteoclasts and those with 2-5 nuclei. Thus, the suppression of CD47 expression could reduce the number of osteoclasts with 2-4 nuclei. Furthermore, the total number of osteoclasts was reduced by the suppression of DC-STAMP and OC-STAMP expression. These results suggest that intermittent continuous mechanical stress is one factor that suppresses the fusion of osteoclasts, and that the cell fusion factor is controlled by the duration and frequency of intermittent stimulation by sustained extension.
Mechanical stress plays an important role in the remodeling of bone. In previous reports by this research team, we showed the suppression of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effects of intermittent 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 10 % for 2 days from the fourth day of culture. In different treatments, mechanical stress was applied for 24 h, followed by bouts of continuous mechanical stress and no stress alternating at 1, 2, 3, 4, 6, or 12 h. In addition, we assayed osteoclast-related genetic mRNA expression using the real-time PCR method. We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts with 2, 3, 4, 5, 6, or 7 nuclei, and those with more nuclei. The number of osteoclasts was lower after mechanical stress when compared with no mechanical stress. The number of total osteoclasts was significantly suppressed in the group stimulated every hour (1-h group) when compared with the group that was stimulated every 12 h (12-h group). Furthermore, we found that the number of osteoclasts with 2-4 nuclei was significantly reduced by intermittent continuous mechanical stress. The measurement of mRNA expression using real-time PCR showed the suppression of DC-STAMP, OC-STAMP, and CD47, which are fusion factors for osteoclasts. These significantly decreased in the 1-h group compared with that in 12-h group. CD47 participates in cell fusion with the mononuclear preosteoclasts among each other, and among the mononuclear preosteoclasts and those with 2-5 nuclei. Thus, the suppression of CD47 expression could reduce the number of osteoclasts with 2-4 nuclei. Furthermore, the total number of osteoclasts was reduced by the suppression of DC-STAMP and OC-STAMP expression. These results suggest that intermittent continuous mechanical stress is one factor that suppresses the fusion of osteoclasts, and that the cell fusion factor is controlled by the duration and frequency of intermittent stimulation by sustained extension.
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