布鲁顿酪氨酸激酶对破骨细胞增殖及分化作用的实验研究

Effects of Bruton’s tyrosine kinase on the proliferation and differentiation 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5d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。

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).Conclusion Silencing of BTK can inhibit the proliferation and differentiation of osteoclasts.BTK can be used as a new target for the inhibition of osteoclasts.