中文摘要：

目的探讨唑来膦酸对破骨细胞分化中Ca^2＋／钙调蛋白依赖性蛋白激酶Ⅱ（Ca^2＋／calmodnlin．dependent protein kinaseⅡ，CaMKⅡ）与钙调蛋结合及下游活化T细胞核因子c1（nuclear factor of activation of cells-1，NFATc1）、抗酒石酸酸性磷酸酶（tartrate resistant acidphos phatase，TRAP）基因表达的影响，探索其抑制破骨细胞分化的分子机制。方法将小鼠RAW264．7细胞分为A、B两组，每组均以5×10^3个／孔接种于直径为30mm的培养皿中培养。A组（对照组）在NF-κB受体激活蛋白配体（receptor activator of NF-κB ligand，RANKL）诱导下向破骨细胞形成，B组（唑来膦酸组）在RANKL诱导培养1d后加用1×10^-6mol／L唑来膦酸处理2d。应用免疫共沉淀co．immunoprecipitation，Co-IP）及反向Co-IP对CaMKⅡ与钙调蛋白的结合进行分析；应用蛋白质印迹法及免疫荧光细胞化学检测两组NFATc1、TRAP蛋白表达，并对破骨细胞生成进行评价。结果B组新生多核破骨细胞数、牙本质吸陷窝数和面积分别为（11．3±1．5）个、（8．7±2．1）个和（5034．4＋775．4）μm^2，均显著低于A组[分别为（37．7±5．7）个、（23．0±4．0）个和（15042．7±1906．0）μm^2]（P〈0．01）。Co—IP及反向Co—IP检测显示，B组CaMKII与钙调蛋白的结合较A组分别显著下降了59．8％和50．9％（P〈0.01）；在总蛋白中，B组钙调蛋白与CaMKll蛋白较A组，分别显著降低了52．1％和51．5％（P〈0．01）。NFATc1、TRAP蛋白水平B组较A组显著下降了52．4％和38．9％（P〈0．01）；免疫荧光化学检测也显示B组蛋白表达强度较A组弱。结论唑来膦酸可以显著抑制破骨细胞分化中CaMKⅡ与钙调蛋白的结合，并下调下游NFATc1、TRAP的蛋白表达。

英文摘要：

Objective To investigate the effect of zoledronate on protein interaction between Ca^2＋/ calmodulin-dependent protein kinase 1/（CaMK Ⅱ ） and ealmodulin and protein expression of nuclear factor of activation of T cells-1 （NFATcl） and tartrate resistant acid phosphatase （TRAP） during osteoelast differentiation. Methods Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand （RANKL） for osteoelastogenesis, and group B was treated with 1 ± 10^-6 zoledronate for two days from day 2. Co-immunoprcipitation （Co-IP） and reverse Co-IP were used to detect the protein-binding between CaMK I] and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc 1 and TRAP in both groups. Osteoclast formation was also analyzed. Results In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3± 1.5, 8.7±2.1 and （5 034.4±775.4） μm^2 respevtively, which were significantly lower than those （37.7＋5.7, 23.0±4.0 and [15 042.7±1 906.0] μm^2 ） in group A （P〈 0.01）. Co-IP and reverse Co-IP examination indicated that protein-binding between CaMK ]/ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A （P〈0.01）. The protein level of calmodulin and CaMK II in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A （P〈0.01）. NFATcl and TRAP protein decreased by 52.4% and 38.9% in group B than in group A （P〈0.01）, respectively. Conclusions Zoledronate could significantly inhibit protein-binding between CaMK 1I and calmodulin and down-regulate protein level of NFATcl and TRAP.