CaMKⅡδ基因沉默对破骨细胞分化功能及c-fos/c-jun/CREB基因的影响  

Effect of CaMKⅡδ gene silencing on osteoclast differentiation and c-fos/c-jun/CREB gene

作　　者：张誉泓 戚孟春 董伟 孙红 Zhang Yuhong;Qi Mengchun;Dong Wei;Sun Hong(Dept.of Oral and Maxillofacial Surgery,College of Stomatology,North China University of Science and Technology,Tangshan 063000,China;Dept.of Pathology,College of Basic Medicine,North China University of Science and Technology,Tangshan 063000,China)

机构地区：[1]华北理工大学口腔医学院口腔颌面外科教研室,唐山063000 [2]华北理工大学基础医学院病理教研室,唐山063000

出　　处：《国际口腔医学杂志》2019年第4期420-425,共6页International Journal of Stomatology

基　　金：国家自然科学基金(81270965);河北省自然科学基金(20-17209114)~~

摘　　要：目的探索编码钙调蛋白依赖性激酶(CaMK)Ⅱδ的基因沉默对破骨细胞分化功能的影响以及c-fos、c-jun、CREB基因在CaMKⅡδ调控破骨细胞分化中的作用,以揭示破骨细胞分化调控的分子机制。方法构建CaMKⅡδ RNA干扰载体,转染至小鼠RAW264.7细胞,确定干扰效率。病毒转染细胞后,用50 ng·mL^-1核因子-κB受体活化因子配体(RANKL)诱导,5 d后收获,通过耐酒石酸酸性磷酸酶(TRAP)检测、牛骨吸收陷窝检测沉默CaMKⅡδ基因对破骨细胞分化、骨吸收功能的影响;48 h后收获,利用实时聚合酶链式反应(PCR)、Westernblot等方法检测c-fos、c-jun、CREB的表达情况。结果 CaMKⅡδ RNA干扰在mRNA和蛋白质水平的干扰效率分别为70.6%和72.2%。 CaMKⅡδ RNA干扰可显著降低 c-fos、c-jun和CREB的mRNA水平,下调幅度分别为74%、49%和24%,CaMKⅡδ RNA干扰可显著降低c-Fos、c-Jun和CREB的水平,下调幅度分别为74.3%、61.3%和59.2%。结论 CaMKⅡδ RNA干扰可在mRNA和蛋白质水平显著抑制c-fos、c-jun、CREB的表达,其共同参与CaMKⅡδ对破骨细胞分化的调控。Objective This study aims to explore the effect of calmodulin-dependent kinase (CaMK)Ⅱδ gene silencing on osteoclast differentiation, investigate the roles of the c-fos, c-jun and CREB genes in CaMKⅡδ regulation of osteoclast differentiation, and reveal the molecular mechanism underlying osteoclast differentiation.Methods A CaMKⅡδ RNA interference vector was constructed and transfected into mouse RAW264.7 cells to determine interference efficiency.The cells were transfected with the virus, induced with 50 ng·mL^-1 nuclear factor-κB receptor activator ligand and then harvested after 5 days.The effect of silencing the CaMKⅡδ gene on osteoclast differentiation was detected by tartrateresistant acid phosphatase detection and bovine bone resorption lacuna.After 48 h, the expression levels of the c-fos, c-jun and CREB genes were detected by real-time polymerase chain reaction and Western blot.Results The interference efficiencies of the CaMKⅡδ RNA interference vector at the mRNA and protein levels were 70.6% and 72.2%, respectively.CaMKⅡδ RNA interference significantly reduced the mRNA levels of c-fos, c-jun and CREB by 74%, 49% and 24%, respectively.CaMKⅡδ RNA interference significantly reduced the protein levels of c-Fos, c-Jun and CREB by 74.3%, 61.3% and 59.2%, respectively.Conclusion CaMKⅡδ RNA interference can significantly inhibit the expression of c-fos, c-jun and CREB at the mRNA and protein levels.These results indicate that c-Fos, c-Jun and CREB are involved in the regulatory effects of CaMKⅡδ on osteoclast differentiation.

关 键 词：破骨细胞 钙调蛋白依赖性激酶Ⅱδ C-FOS C-JUN 环腺苷酸反应原件结合蛋白 

分 类 号：Q786[生物学—分子生物学]