唑来膦酸影响单个核细胞向破骨细胞分化及RANK表达的研究

Effects of zoledronate on RANK expression and differentiation of mononuclear cells to osteoclasts

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 investigate the effect of zoledronate on osteoclastogenesis and theexpression of Capthsin K, Receptor activator of nuclear factor kappa-B（RANK） which related with Osteoclast differentiation, and explore the mechanism of action of zoledronate on osteoclastogenesis. Methods The receptor activator of nuclear factor kappa-B ligand （RANKL） was used to induce differentiation of RAW264.7 cells into osteoclasts in vitro. The cells were divided into two groups at the beginning of the induction：Group one（ G1 ） were induced by 50 ng/ml RANKL as the control group;Group two（G2） were treated with 1 × 10^-6 mol/L zoledronate simultaneously with the osteoclast induction. The cells were acquired after four days induction. The cells were stained with tartrate resistant acid phosphatase （TRAP） and the osteoclastswere counted. Activity of alkaline phosphatase was detected. The expression of Cathepsin K and RANK were detected by Real-time PCR and western blot, respectively. Results The osteoclastogenesis was decreased by the addition of zoledronate. The number of TRAP ＋ cells in G2 was decreased（62.0 ± 10.2）% as compared to GI （P 〈0.05）. TRAP activity in G2 was declined as （ 31.0 ± 4.1 ） % as compared to G1 （ P 〈 0.05 ） ; the expression of Cathepsin K and RANK in G2 were significant declined by （78.0 ±4.2）% and （49.0±1. 9）% when compared to G1 （P 〈0.05）. Conclusion Zoledronate can decrease osteoclastogenesis by inhibiting the expression of RANK in osteoclast precursors.