Objective: To observe the effect of Bruton's tyrosine kinase (BTK) on the proliferation and differentiation of osteoclasts and to explore the mechanism of BTK on bone destruction in periapical periodontitis.
Methods: After RAW264.7 cells induced with 100 ng·L⁻¹ receptor activator for nuclear factor-κB ligand (RANKL) for 5 days, osteoclast induction was confirmed by light microscopy, tartrate-resistant acid phosphatase (TRAP) staining, and quantitative real-time PCR (RT-qPCR). Then, BTK-small interfering RNA (BTK-siRNA) was transfected into cells induced for 5 days. After 24 h, the expression of TRAP mRNA was measured using RT-qPCR, and the proliferation and differentiation of osteoclasts were detected using CCK-8 and TRAP activity assay. Statistical analysis was performed.
Results: After RAW264.7 was induced with RANKL for 5 days, a large number of round, ellipse, irregularly protuberant, and TRAP-positive macrophages were observed under light microscopy. The expression of TRAP mRNA significantly reduced after 24 h of BTK-siRNA transfection (P<0.05). The detection of CCK-8 and TRAP activities showed that the proliferation and differentiation of osteoclasts significantly decreased (P<0.05).
Conclusions: Silencing of BTK can inhibit the proliferation and differentiation of osteoclasts. BTK can be used as a new target for the inhibition of osteoclasts.
目的 观察布鲁顿酪氨酸激酶（BTK）对破骨细胞增殖及分化的影响，探讨BTK对根尖周炎骨破坏的作用。方法 破骨前体细胞RAW264.7经100 ng·L⁻¹核因子κB受体活化因子配体（RANKL）诱导5 d后，通过观察细胞形态、抗酒石酸酸性磷酸酶（TRAP）染色及实时荧光定量PCR（RT-qPCR）的方法，验证破骨细胞是否诱导成功。破骨细胞诱导成功后，转染BTK-小干扰RNA（siRNA）24 h，利用RT-qPCR检测TRAP mRNA的表达，采用CCK-8和TRAP酶活性检测法检测破骨细胞的增殖及分化情况。结果 RAW264.7细胞经RANKL诱导5 d后，可见大量体积较大，外观呈圆形、椭圆形，周围有不规则突起及TRAP染色阳性的多核破骨细胞。经BTK-siRNA转染24 h后，破骨细胞TRAP mRNA的表达水平显著降低（P<0.05），其增殖及分化能力也明显受到抑制（P<0.05）。结论 抑制BTK表达，可以抑制破骨细胞的增殖及分化；BTK可作为抑制破骨细胞的新靶点。.
Keywords: Bruton’s tyrosine kinase; bone destruction; osteoclast; small interfering RNA transfection.