DNAX相关蛋白12信号通路在压应力诱导小鼠单核细胞向破骨细胞分化中的作用  

Effects of DNAX-associated protein 12 signal pathways on differentiation of mouse monocytes RAW264. 7 into osteoclasts by tensile strain

作　　者：黄生高[1] 凌天牖[1] 钟孝欢[1] 熊祎[1] 刘云峰[1] 梁富英[1] 

机构地区：[1]中南大学湘雅二医院口腔医疗中心,长沙410011

出　　处：《中华口腔医学杂志》2012年第9期562-566,共5页Chinese Journal of Stomatology

摘　　要：目的探讨DNAX相关蛋白12（DNAX—associatedprotein12，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，构建并导人DAPl2短发夹RNA（shorthairpinRNA，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DAPl2、抗酒石酸酸性磷酸酶（tartrate-resistantacidphosphataseTRAP）、非受体酪氨酸激酶家族Btk和Tec激酶、活化T细胞核因子1（nuclearfactorofactivatedTcells1，NFATcl）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，DAPl2．shRNA干扰组DAPl2mRNA表达水平（0．112±0．025）明显降低（P〈0．05）；DAP12-shRNA干扰组DAPl2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0h（0．497±0．031）相比，加力6h（0．671±0．031）和加力12h（0．800±0．043）TRAPmRNA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、12h）（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、12h）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 mon0eytes 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.

关 键 词：基因沉默 应力 物理 单核细胞 DAP12信号通路 

分 类 号：R780.2[医药卫生—口腔医学]