Permalink : http://hdl.handle.net/2115/86833

KEYWORDS : RAW264.7細胞;破骨細胞;継代数;RAW264.7cell;osteoclast;passage number

歯科矯正治療による歯の移動は，メカニカルストレスに対する歯周組織の反応として捉えることができる．歯周組織に矯正力が加わることにより，圧迫側では骨添加の抑制と骨吸収の促進が，牽引側では骨吸収の抑制と骨添加の促進が起こり，骨のリモデリングが生じる． 細胞に直接的に機械的刺激を加えることで，RAW264.7（RAW）細胞から破骨細胞への分化誘導系に与える影響を調べようとしたところ，RAW細胞の継代数により破骨細胞の分化・誘導に違いが生じた．RAW細胞の継代数と破骨細胞の分化誘導系の関連を検討した報告は未だないため，本研究で検討した．継代数が10未満であるN10RAW細胞と継代数が30以上であるN30RAW細胞に対して破骨細胞誘導培地を用いて７日間培養し，破骨細胞数の推移を観察した．N10RAW細胞から分化したN10破骨細胞は4 日目をピークとして増加し，その後減少した．N30RAW細胞から分化したN30破骨細胞は6 日目をピークとして増加し，その後減少した．次に細胞増殖試験を行った結果，N10RAW細胞とN30RAW細胞に有意差を認めなかった．破骨細胞関連遺伝子であるTRAP，RANK，CD47，NFATc1，DC-STAMP，OC-STAMP，IntegrinαⅤ，IntegrinβⅢ，CD11a，CD11bの発現量の差を調査するために，Real time RT-PCR 法を行った．培養3日目ではRANK，NFATc1，IntegrinβⅢでは有意差を認めなかったが，細胞融合因子であるCD47，DC-STAMP，OC-STAMP，IntegrinαⅤにおいて，N10破骨細胞と比較してN30破骨細胞では有意に減少した．これらの結果から培養3 日目にN10破骨細胞と比較してN30破骨細胞では破骨細胞の細胞融合が抑制されることが示唆された．以上の結果から，破骨細胞への分化が早いN10破骨細胞と分化が遅いN30破骨細胞を実験に用いる際には，それぞれの特性を活かして用いなければならないことが示唆された．
Teeth movement after orthodontic treatment can be regarded as t he reaction of periodontal tissue to mechanical stress. By applying orthodontic force to the periodontium, bone addition is suppressed and bone resorption is promoted on the compression side. Furthermore, bone resorption is suppressed and bone addition is promoted on the traction side, resulting in bone remodeling. When we investigated the effect of RAW 264.7 (RAW) cells on the osteoclast differentiation-inducing system by directly applying mechanical stimulation to the cells, we showed the differentiation of osteoclasts by the number of passages of RAW cells. Additionally, there was a difference in induction. Therefore, in this study, we assessed the relationship between the passage number of RAW cells and the differentiation-inducing system of osteoclasts as there are no available reports in this regard. N10 R AW cells having a passage number <10 and N30 R AW cells having a passage number ¬30 were cultured for seven days in an osteoclast-inducing mediu m, and changes in the number of osteoclasts were observed. N10 osteoclasts differentiated from N10 RAW cells increased after peaking on the day 4 and then decreased. Similarly, N30 osteoclasts differentiated from N30 RAW cells increased after peaking on day 6 and then decreased. Next, as a result of the cell proliferation test, no significant difference was observed between N10 and N30 RAW cells. Real-time polymerase chain reaction (RT-PCR) was performed to investigate the differences in the expression levels of osteoclast-related genes including TRAP, RANK, CD47, NFATc1, DC-STAMP, OC-STAMP, Integrin αV, IntegrinβⅢ, CD11a, and CD11b. No significant difference was observed in RANK, NFATc1, and Integrin βⅢ expression levels on the day 3of culture. In contrast, it decreased signif icantly in the cell fusion factors (CD47, DC-STAMP, OC-STAMP, and Integrinα V) in N30 osteoclasts compared with those in N10 osteoclasts. These results suggest that cell fusion of osteoclasts is suppressed in N30 osteoclasts compared with N10 osteoclasts on day 3 of culture. The above results suggested that when using N10 osteoclasts, which differentiate rapidly into osteoclasts, and N30 osteoclasts, which differentiate slowly into osteoclasts, it is necessary to take advantage of their characteristics.