中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2012年
9期
562-566
,共5页
黄生高%凌天牖%钟孝欢%熊祎%刘云峰%梁富英
黃生高%凌天牖%鐘孝歡%熊祎%劉雲峰%樑富英
황생고%릉천유%종효환%웅의%류운봉%량부영
基因沉默%应力,物理%单核细胞%DAP12信号通路
基因沉默%應力,物理%單覈細胞%DAP12信號通路
기인침묵%응력,물리%단핵세포%DAP12신호통로
Gene silencing%Stress,mechanical%Monocytes%DAP12 signal pathway
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.
目的 探討DNAX相關蛋白12( DNAX-associated protein 12,DAP12)信號通路在壓應力誘導小鼠單覈細胞嚮破骨細胞分化過程中的作用,以期闡明正畸治療過程中牙槽骨破骨細胞分化的調節機製.方法 以小鼠單覈細胞RAW264.7為研究對象,構建併導入DAP12短髮夾RNA(short hairpin RNA,shRNA)質粒,分為DAP12-shRNA榦擾組、陰性對照組和空白對照組,採用四點彎麯體外細胞加載裝置加載壓應力,以反轉錄聚閤酶鏈反應、蛋白質印跡法分彆檢測DAP12、抗酒石痠痠性燐痠酶(tartrate-resistant acid phosphatase TRAP)、非受體酪氨痠激酶傢族Btk和Tec激酶、活化T細胞覈因子1( nuclear factor of activated T cells 1,NFATc1)mRNA和蛋白的錶達情況.結果 與陰性對照組(0.385 ±0.021)和空白對照組(0.391 ±0.009)相比,DAP12-shRNA榦擾組DAP12 mRNA錶達水平(0.112 ±0.025)明顯降低(P＜0.05)；DAP12-shRNA榦擾組DAP12蛋白錶達(0.193 ±0.015)顯著低于陰性對照組(0.526±0.006)和空白對照組(0.514±0.024) (P ＜0.05).榦擾組內與壓應力刺激細胞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錶達顯著增加(P＜0.05),但各對應時間點的錶達均比陰性對照組弱(3、6、12 h)(P ＜0.05).受壓應力刺激DAP12-shRNA榦擾組Btk、Tec、NFATc1的錶達雖然隨加力時間的增加而增加,但各時間點(6、12 h)與陰性對照組比均顯著減少(P＜0.05).結論 DAP12信號通路存在併參與調節壓應力刺激誘導的破骨細胞分化,DAP12在其中起重要作用.
목적 탐토DNAX상관단백12( DNAX-associated protein 12,DAP12)신호통로재압응력유도소서단핵세포향파골세포분화과정중적작용,이기천명정기치료과정중아조골파골세포분화적조절궤제.방법 이소서단핵세포RAW264.7위연구대상,구건병도입DAP12단발협RNA(short hairpin RNA,shRNA)질립,분위DAP12-shRNA간우조、음성대조조화공백대조조,채용사점만곡체외세포가재장치가재압응력,이반전록취합매련반응、단백질인적법분별검측DAP12、항주석산산성린산매(tartrate-resistant acid phosphatase TRAP)、비수체락안산격매가족Btk화Tec격매、활화T세포핵인자1( nuclear factor of activated T cells 1,NFATc1)mRNA화단백적표체정황.결과 여음성대조조(0.385 ±0.021)화공백대조조(0.391 ±0.009)상비,DAP12-shRNA간우조DAP12 mRNA표체수평(0.112 ±0.025)명현강저(P＜0.05)；DAP12-shRNA간우조DAP12단백표체(0.193 ±0.015)현저저우음성대조조(0.526±0.006)화공백대조조(0.514±0.024) (P ＜0.05).간우조내여압응력자격세포0 h(0.497 ±0.031)상비,가력6 h(0.671±0.031)화가력12 h(0.800±0.043) TRAP mRNA표체현저증가(P＜0.05),단각대응시간점적표체균비음성대조조약(3、6、12 h)(P ＜0.05).수압응력자격DAP12-shRNA간우조Btk、Tec、NFATc1적표체수연수가력시간적증가이증가,단각시간점(6、12 h)여음성대조조비균현저감소(P＜0.05).결론 DAP12신호통로존재병삼여조절압응력자격유도적파골세포분화,DAP12재기중기중요작용.
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.
