双膦酸盐（BPs）通过抑制破骨细胞（OC）治疗骨过度吸收性疾病，作用机制远未弄清，制约了临床应用及新药开发。Ca2+/calmodulin/NFATc1信号通路对OC生成至关重要；但是否参与BPs细胞效应尚不清楚。我们前期研究发现，阿伦膦酸(ALN)使OC[Ca2+]i显著上升，NFATc1表达下调，并抑制OC生成；Ca2+通道拮抗剂则逆转了上述效应；提示Ca2+/calmodulin/NFATc1通路可能参与其中。为了证实该假设，本课题拟：①进一步对BPs作用下OC[Ca2+]i及Ca2+波动、calmodulin、NFATc1等基因表达进行检测，明确信号分子间联动关系；②应用酶活性分析及蛋白结合试验，评价信号蛋白活性及相互作用；③采用RNA干扰、基因重组技术证实上述分子在BPs信号传导中的关键作用。以进一步阐明BPs分子机制；为开发新的OC靶向药物，治疗骨过度吸收疾病提供理论依据。

Bisphosphonates (BPs) are widely used for treatment of many bone diseases involving excessive bone resorption and they act mainly through inhibiting the formation and function of osteoclasts. However,the molecular mechanisms of BPs remains far less understood and this contricts their further clinical application and development of new drugs for treatment of above diseases..Ca2+/calmodulin/NFATc1(neclear factor of activated T cells c 1) signaling pathway is essential for differentiation and formation of osteoclast, while its function in BPs-induced inhibition of osteoclastogenesis remains unknown. Recently, we found alendronate (ALN), a third generation of BPs, could significantly downregulate gene expression of NFATc1 and inhibit osteoclastogenesis of RAW264.7 cells. The above effects of ALN was partly inverted by Gd3+, an antagonist of Ca2+ channel for control of Ca2+ influx. based on above results, we hypothesize that Ca2+/calmodulin/NFATc1 signaling pathway may play a key role in the intracellular action of ALN. To prove the hypothesis: ①The influences of ALN on [Ca2+]i, oscillation of Ca2+, activation of NFATc1, as well as gene expression of calmodulin, calcineurin, CaMKII and CaMKIV, are to be studied. The relationships among the molecules are also to be explored. ② Protein activity analysis and protein-binding experiment are to be performed to verify the activity and interaction of the molecules. ③ RNA interference and gene recombinant technique are also used to verify the function of these molecules on ALN-induced inhibition of osteoclastogenesis. The results of this study will broaden our knowledge on molecular mechanisms of BPs and benefit the development of new, more potent, osteoclast-targeting drugs for treatment of diseases invovling excessive bone resorption.