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目的研究唑来膦酸(zoledronate,ZLN)对破骨细胞分化及相关信号分子组织蛋白酶K(capthsin K)、细胞核因子κB受体活化因子(receptor activator of nuclear factor kappa-B,RANK)表达的影响,初步探讨唑来膦酸对破骨细胞分化的影响和机制。方法采用核因子κB受体活化因子配体(receptor activator of nuclear factor kappa-B ligand,RANKL)诱导小鼠单核巨噬细胞株RAW264.7向破骨细胞分化。在诱导开始时即分成2组:G1组为空白对照组,采用50 ng/ml RANKL诱导;G2组为实验组,从诱导开始就加入1×10~(-6)mol/L唑来膦酸处理,诱导4 d后收获并进行抗酒石酸磷酸酶(TRAP)染色观察、计算破骨细胞数量并检测TRAP酶活性;采用Real-Time PCR和Western Blot分析组织蛋白酶K、RANK的表达水平。结果唑来膦酸组生成的TRAP阳性的破骨样细胞较对照组少且核的数目也较少,唑来膦酸组较对照组破骨细胞生成的数目下降[(62.0±10.2)%,P<0.05];唑来膦酸组TRAP活性较对照组下降[(31.0±4.1)%,P<0.05];唑来膦酸组的组织蛋白酶K、RANK基因表达较对照组分别下降[(78.0±4.2)%、(49.0±1.9)%,P<0.05]。结论唑来膦酸通过抑制破骨前体细胞RANK的表达来抑制体外破骨细胞分化。
Objective To study the effect of zoledronate (ZLN) on the differentiation of osteoclasts and the expression of capthsin K and receptor activator of nuclear factor kappa-B (RANK) , Preliminary study of zoledronic acid on osteoclast differentiation and mechanism. Methods The RAW264.7 cells were induced to differentiate into osteoclasts by using receptor activator of nuclear factor kappa-B ligand (RANKL). At the beginning of induction, animals were divided into 2 groups: G1 group was blank control group, which was induced by 50 ng / ml RANKL; G2 group was experimental group, 1 × 10 -6 mol / L zoledronic acid The cells were harvested and treated with TRAP for 4 days. The number of osteoclasts and the activity of TRAP were measured. The expression of cathepsin K and RANK was analyzed by Real-Time PCR and Western Blot. Results The numbers of TRAP positive osteoclast-like cells produced by zoledronic acid group were fewer and the number of nuclei was less than that of the control group. The number of osteoclasts in zoledronic acid group was decreased by 62.0 ± 10.2% P <0.05]. The activity of TRAP in zoledronic acid group was lower than that in control group [(31.0 ± 4.1)%, P <0.05], while the expression of cathepsin K and RANK gene in zoledronic acid group was decreased ± 4.2%, (49.0 ± 1.9)%, P <0.05]. Conclusion Zoledronic acid inhibits osteoclast differentiation in vitro by inhibiting the expression of RANK in osteoclast precursors.