唑来膦酸对多发性骨髓瘤成骨细胞增殖及其RANKL/OPG表达的影响

目的研究唑来膦酸对多发性骨髓瘤(MM)患者成骨细胞(OB)增殖及其细胞核因子κ-B受体活化因子配体(RANKL)和护骨素(OPG)表达的影响。方法取MM患者骨髓液密度梯度离心法分离单个核细胞,原代培养,贴壁传代后加入含维生素C、地塞米松、β-甘油磷酸钠的条件培养基,诱导成OB,采用不同浓度唑来膦酸处理培养的OB,分别作用24、48和72 h后,通过①细胞计数试剂盒(CCK-8)检测OB增殖;②RT-PCR检测OB RANKL和OPG mRNA表达;和③酶联免疫吸附实验(ELISA)检测相应细胞培养上清液中sRANKL和OPG浓度来考察唑来膦酸的作用。结果①OB用唑来膦酸处理后,OB的CCK-8实验吸光值呈不同程度升高。②唑来膦酸作用24 h和48 h后,OB RANKLmRNA表达变化不明显。作用72 h,低浓度(10-9 mol.L-1)唑来膦酸显示能降低RANKL mRNA表达(P<0.05)。OB培养上清的sRANKL浓度也呈现类似的变化。③唑来膦酸作用24 h后,OB OPG mRNA表达变化不明显。作用48h,呈不同程度增加,以10-8 mol.L-1浓度最明显(P<0.05)。作用72 h,呈不同程度增加,以10-7 mol.L-1浓度最明显(P<0.05)。结论唑来膦酸有促进OB增殖的作用;唑来膦酸可能下调OB RANKL表达,并且上调OPG表达。

Effects of zoledronic acid on osteoblasts proliferation and their RANKL/OPG expression in multiple myeloma patients

Aim To investigate the effect of zoledronic acid on osteoblasts(OB)proliferation and their expression of receptor activator of nuclear factor κ-B ligand(RANKL) and osteoprotegerin(OPG) in multiple myeloma(MM) patients.Methods Bone marrow mononuclear cells were isolated from MM patients with density gradient centrifugation.Adherent cells were collected and cultured.Passage cells were incubated in condition medium containing dexamethasone,vitamin C,and β-glycerophosphate.The cells were harvested and assumed to be OB.They were treated with Zoledronic acid at different concentration for 24,48 and 72 h.Cell counting kit-8(CCK-8) was used to evaluate cell proliferation.RT-PCR was used for expression of RANKL/OPG mRNA of OB and enzyme linked immunosorbent assay(ELISA) for soluble RANKL(sRANKL)/OPG in cell culture fluid.Results ① Zoledronic acid increased absorbance values in OB CCK-8 assay.② There was no significant change in OB RANKL mRNA expression after 24 h and 48 h zoledronic acid treatment.After 72 h treatment,expression decreased,especially at the concentration of zoledronic acid of 10-9 mol·L-1(P<0.05).③ No significant change was found in OB OPG mRNA expression after 24 h zoledronic acid treatment.It increased after 48 h and 72 h treatment,especially at the concentration of 10-8 mol·L-1 and 10-7 mol·L-1(P<0.05).Conclusion Zoledronic acid might increase OB proliferation,down-regulate their RANKL expression and up-regulate their OPG expression.