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

摘要

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 CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264. 7 cells. The experiment was di-vided into three groups:A,B and C,which were the control group,negative vector group and interference vector group. After transfec-tion for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days. Real-time quanti-tative 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% ,re-spectively(P0. 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,sugges-ting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.%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 re-ceptor 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CaMKIIγ RNA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% (P0. 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 CaMKIIγ RNA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.