阿仑膦酸钠联合骨化三醇对成骨细胞的调节及对OPG/RANKL蛋白表达的影响

The regulatory effects of alendronate combination with calcitriol on osteoblast and the expression profile of OPG/RANKL proteins

目的探讨阿仑膦酸钠联合骨化三醇对成骨细胞碱性磷酸酶(alkaline phosphatase,AKP)活性、细胞增殖及OPG/RANKL蛋白表达的影响。方法以骨髓间充质干细胞诱导分化成骨细胞,获得成骨细胞后分别加入不同浓度阿仑膦酸钠(1×10^(-10)、1×10^(-9)、1×10^(-8)、1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4) mmol/L)与骨化三醇(1×10^(-8)、1×10^(-7)、1×10^(-6)、1×10^(-5)、1×10^(-4)、1×10^(-3)、1×10^(-2) mmol/L),测定成骨细胞AKP活性及观察茜素红染色情况,继而确定最佳单药浓度。比较两种单药及联合用药(均选择最佳单药浓度)对成骨细胞AKP活性、细胞增殖及OPG/RANKL蛋白表达的影响。多组间AKP活性的比较采用方差分析,进一两组间的比较采用q检验,率的比较采用χ2检验。结果成骨细胞诱导分化成功。阿仑膦酸钠与骨化三醇的最佳单药浓度分别为1×10^(-6)mmol/L、1×10^(-4) mmol/L。相对于无药物处理的成骨细胞组,各药物处理组差异明显,其中阿仑膦酸钠+骨化三醇的联合用药组成骨细胞AKP活性最强、钙结节形成最多、细胞增殖最明显,其次为骨化三醇单药组,再次为阿仑膦酸钠组;另外,单纯成骨细胞诱导组及联合用药组的OPG蛋白表达均明显升高、RANKL蛋白表达明显降低,而且联合用药组较单纯成骨细胞诱导组OPG蛋白的升高水平更显著、RANKL蛋白的下降水平更显著。结论阿仑膦酸钠联合骨化三醇具有显著的调节成骨细胞AKP活性、钙结节形成、细胞增殖及OPG/RANKL蛋白表达的作用。

Objective This study is to investigate alkaline phosphatase activity,calcium nodules formation and proliferation of alendronate combination with calcitriol on osteoblast.Methods Bone marrow mesenchymal stem cells were differentiated into osteoblast.Different concentration of alendronate(1×10^(-10),1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6),1×10^(-5),and 1×10^(-4) mmol/L)and calcitriol(1×10^(-8),1×10^(-7),1×10^(-6),1×10^(-5),1×10^(-4),1×10^(-3),and 1×10^(-2) mmol/L)were added,and alkaline phosphatase(AKP)activity and calcium nodules formation were determined.The effect of mono intervention and combined intervention on AKP activity,calcium nodules formation,proliferation,and protein expression levels of OPG/RANKL were compared.Comparison of AKP activity among groups were applied by ANOVA followed by q test.Ratio comparison was achieved byχ2 test.Results Osteoblast was successfully differentiated.Alendronate of 1×10^(-6)mmol/L and calcitriol of 1×10^(-4)mmol/L were the best concentration for osteoblast differentiation.Compared with control group or osteoinduction group,the alkaline phosphatase activity and alizarin red staining level have been significantly improved in combination therapy group.Combination therapy of alendronate and calcitriol can significantly promote proliferation,increase the expression level of OPG,and decrease the expression level of RANKL.These effects in combination were better than calcitriol and alendronate alone.Conclusion The combination therapy of alendronate and calcitriol has significant regulatory effects on alkaline phosphatase activity,calcium nodules formation,proliferation of osteoblast,which is associated with the protein expression profile of OPG/RANKL.