检索历史 清除

目的 探讨DNAX相关蛋白12( DNAX-associated protein 12,DAP12)信号通路在压应力诱导小鼠单核细胞向破骨细胞分化过程中的作用,以期阐明正畸治疗过程中牙槽骨破骨细胞分化的调节机制.方法 以小鼠单核细胞RAW264.7为研究对象,构建并导入DAP12短发夹RNA(short hairpin RNA,shRNA)质粒,分为DAP12-shRNA干扰组、阴性对照组和空白对照组,采用四点弯曲体外细胞加载装置加载压应力,以反转录聚合酶链反应、蛋白质印迹法分别检测DAP12、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase TRAP)、非受体酪氨酸激酶家族Btk和Tec激酶、活化T细胞核因子1( nuclear factor of activated T cells 1,NFATc1)mRNA和蛋白的表达情况.结果 与阴性对照组(0.385 ±0.021)和空白对照组(0.391 ±0.009)相比,DAP12-shRNA干扰组DAP12 mRNA表达水平(0.112 ±0.025)明显降低(P＜0.05)；DAP12-shRNA干扰组DAP12蛋白表达(0.193 ±0.015)显著低于阴性对照组(0.526±0.006)和空白对照组(0.514±0.024) (P ＜0.05).干扰组内与压应力刺激细胞0 h(0.497 ±0.031)相比,加力6 h(0.671±0.031)和加力12 h(0.800±0.043) TRAP mRNA表达显著增加(P＜0.05),但各对应时间点的表达均比阴性对照组弱(3、6、12 h)(P ＜0.05).受压应力刺激DAP12-shRNA干扰组Btk、Tec、NFATc1的表达虽然随加力时间的增加而增加,但各时间点(6、12 h)与阴性对照组比均显著减少(P＜0.05).结论 DAP12信号通路存在并参与调节压应力刺激诱导的破骨细胞分化,DAP12在其中起重要作用.

Objective To explore the effect of DNAX-associated protein 12(DAP12) pathway on the transformation from mouse monocytes RAW264.7 to osteoclasts induced by tensile strain.Methods DAP12shRNA plasmid was constructed and introduced to RAW264.7 cells.Then we supplied tensile strain to RAW264.7 cells by four-point bending system. The mRNA or protein expression of DAP12,tartrate-resistant acid phosphatase(TRAP),tyrosine kinases Btk and Tec and nuclear facior of activated T cells 1 (NFATc 1 ) was measured by reverse transcription PCR (RT-PCR) and Western blotting respectively.Results The expression of DAP12 mRNA (0.112 ± 0.025 ) and protein (0.193 ± 0.015 ) both declined sharply after plasmid being introduced into monocytes RAW264.7 ( P ＜ 0.05 ). After silencing DAP12 expression in RAW264.7 cells by RNA interference,tensile strain-induced TRAP mRNA expression of RAW264.7 cells increased at 6 h(0.671 ±0.031) and 12 h(0.800±0.043) (P＜0.05),but it was weaker than non-RNA-interference-groups at each time point(P ＜0.05).After silencing DAP12 expression in RAW264.7 cells by RNA interference,the expressions of Btk,Tec,NFATcl increased as time passed (6,12 h) ( P ＜ 0.05 ),but the expressions on corresponding time decreased sharply compared with those in control groups ( P ＜ 0.05 ).Conclusions DAP12 pathway play an important role in regulating osteoclast differentiation induced by tensile strain.