高浓度唑来膦酸对人成骨细胞成骨分化及凋亡影响的实验研究

High concentration of zoledronate inhibits osteogenesis differentiation and promotes apoptosis of human osteoblasts

目的:研究高浓度唑来膦酸对体外分离的健康人下颌骨成骨细胞的影响。方法 :体外分离培养来源于健康人下颌骨的成骨细胞,取P3代成骨细胞,用1μmol/L、5μmol/L唑来膦酸分别处理,不加药组为空白对照。以CCK8检测细胞增殖,流式细胞技术检测细胞凋亡,ALP染色及定量分析检测细胞成骨分化,荧光定量PCR检测成骨相关基因ALP、OPN和Runx2的表达,Western免疫印迹检测OPN蛋白的表达。采用SPSS16.0软件包对数据进行统计学分析。结果:唑来膦酸处理后的成骨细胞增殖减慢,5μmol/L处理组较1μmol/L组增殖更慢。药物处理后的细胞凋亡较对照组增加,且随着药物浓度的增加,凋亡百分比显著上升。1μmol/L及5μmol/L唑来膦酸处理后的成骨细胞的成骨分化能力较对照组显著下降。荧光定量PCR结果提示,药物处理后的成骨相关基因ALP、OPN和Runx2的表达显著降低。Western免疫印迹检测结果显示药物处理组OPN蛋白表达量显著下降。结论:高浓度唑来膦酸抑制来源于人下颌骨成骨细胞的增殖及成骨分化,促进其凋亡发生。

PURPOSE： To investigate the influence of zoledronate on osteoblasts isolated from human jaw in vitro.METHODS： Osteoblasts were isolated from health human mandible. Third passage of osteoblasts were exposed to zoledronate with different concentrations（0 μmol/L,1 μmol/L,5 μmol/L） in vitro. Cell proliferation ability was detected using CCK-8 kit and flow cytometry method was applied to identity cell apoptosis. ALP staining and quantitative analysis were used to evaluate the osteogenesis differentiation of osteoblasts. Real-time PCR were used to measure the gene expression of ALP,OPN and Runx2 and western blot（WB） was used to detect OPN expression.The difference between each group was analyzed with SPSS 16.0 software package. RESULTS： Cel l growth curve indicated that zoledronate treatment groups showed slower growth compared with control group and flow cytometry results indicated higher proportion of apoptosis cells in zoledronate treated groups. ALP staining and quantitative analysis both revealed decreased osteogenesis differentiation ability in zoledronate treated groups. These effect were more obvious in 5 μmol/L treated group than 1μmol/L treated group.Significant down-regulation of of ALP, Runx2 and OPN gene expression were also found in zoledronate treated groups.WB results revealed the decreased expression of OPN in zoledronate treated groups. CONCLUSION： High concentration of zoledronate inhibits osteogenesis differentiation and proliferation ability of human osteoblasts and facilitate their apoptosis.