CaMKⅡγ基因沉默对破骨细胞分化中NFATc1、TRAP、c-Src基因表达的影响  

Effect of CaMKⅡγ RNA interference on the expression of NFATc1,TRAP and c-Src genes in osteoclast differentiation

作　　者：刘梦楠 王会 戚孟春 董伟 李任 孙红 LIU Mengnan;WANG Hui;QI Mengchun;DONG Wei;LI Ren;SUN Hong(Department of Oral and Maxillofacial Surgery,College of Stomatology,North China University of Science and Technology,Tangshan 063000,China;Department of Pathology,College of Basic Medicine,North China University of Science and Technology)

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

出　　处：《实用肿瘤学杂志》2019年第4期294-299,共6页Practical Oncology Journal

基　　金：国家自然科学基金(编号:81270965);河北省教育厅重点研究项目(编号:ZD2015005);河北省自然科学基金(编号:H2017209114)

摘　　要：目的研究钙调蛋白依赖性激酶Ⅱ(Calmodulin-dependent kinase Ⅱ,CaMKⅡ)γ基因沉默对破骨细胞分化过程中活化T-细胞核因子c1(Nuclear factor of activated T-cells cytoplasmic 1,NFATc1)、非受体酪氨酸激酶(Cell-sarcoma receptor coactivator,c-Src)、抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)基因表达的影响,探讨CaMKⅡγ在破骨细胞分化中的作用和分子机制。方法用慢病毒构建CaMKⅡγRNA干扰载体,转染RAW264.7细胞,实验分为A、B、C三组,分别为对照组、阴性载体组和干扰载体组;三组细胞转染12 h后,用50 ng RANKL诱导向破骨细胞分化;诱导5 d后收获细胞,采用实时荧光定量PCR、Western blot、免疫荧光化学检测三组细胞中NFATc1、TRAP、c-Src基因表达情况。结果 C组中NFATc1、TRAP、c-Src mRNA水平较A组分别下降了49.86%、43.65%和53.57%(P<0.001),蛋白水平分别下降了54.22%、46.75%和45.86%(P<0.001);而B组与A组比较无统计学差异(P>0.05)。免疫荧光化学检测C组上述基因荧光强度明显弱于A、B组,且破骨细胞生成明显少于A、B组。结论 CaMKⅡγRNA干扰显著抑制了NFATc1、TRAP、c-Src基因表达,提示CaMKⅡγ在破骨细胞分化中起着关键调控作用。Objective The aim of this study was to investigate the effect of Ca2+/calmodulin-dependent kinase Ⅱ(CaMKⅡ)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation.Methods The CaMKⅡγ RNA interference vector was constructed by lentivirus and transfected into RAW264.7 cells.The experiment was divided into three groups:A,B and C,which were the control group,negative vector group and interference vector group.After transfection for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days.Real-time quantitative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups.Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49.86%,43.65% and 53.57%,respectively(P<0.001),and the protein levels decreased by 54.22%,46.75% and 45.86%,respectively(P<0.001).There was no significant difference between the A and the B groups(P>0.05).The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups.Conclusion CaMKⅡγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,suggesting that CaMKⅡγ plays a key regulatory role in osteoclast differentiation.

关 键 词：钙调蛋白依赖性激酶Ⅱγ RNA干扰 活化T细胞核因子c1 非受体酪氨酸激酶 抗酒石酸酸性磷酸酶 

分 类 号：R782[医药卫生—口腔医学]