帕米磷酸钠对大鼠成骨细胞RANKL/OPG mRNA表达的影响

Effect of bisphosphonates pamidronate on the expression of RANKL/OPG mRNA in primary rat osteoblasts

目的观察帕米膦酸钠对新生大鼠成骨细胞核因子κβ受体活化受体配体(RANKL)/护骨素(OPG)mRNA表达的影响。方法体外分离培养新生大鼠成骨细胞,分别观察不同浓度帕米膦酸钠或用帕米膦酸钠处理不同时间对成骨细胞RANKL/OPGmRNA表达的影响。采用半定量RT-PCR方法检测RANKL/OPGmRNA表达。结果帕米膦酸钠呈浓度依赖性降低RANKLmRNA的表达,干预24h,10-5M抑制作用最大,约为对照组表达量的40%(P<0.01)。同时帕米膦酸钠呈浓度依赖性增加OPGmRNA表达,10-6M时增加最明显,约为对照组表达量的2.2倍(P<0.01),而在10-5M时又明显降低。10-6M帕米膦酸钠呈时间依赖性抑制RANKLmRNA和促进OPGmRNA表达,分别于24h抑制RANKLmRNA表达、48h促进OPGmRNA表达且达最大效应(P<0.01)。结论帕米膦酸钠可能通过调节成骨细胞RANKL/OPG的基因表达,而抑制破骨细胞介导的骨吸收。

Objective To observe the effect of bisphosphonates pemidronate on the expression of RANKL and osteoprotegerin（OPG） mRNA in primary rat osteoblasts. Methods Primary osteoblastic ceils were obtained from the calvaria of newborn SD rats by digestion with 0. 1% collagenase. The ceils were treated with pemidronate according to the experimental design. Total cellular RNA was isolated with Trizol. Semiquantitative reverse transcription RCR method was used to determine the level of RANKL and OPG mRNA. Results Pamidronate dose-dependently inhibited RANKL mRNA levels with a maximum effect of 60% after 24 h of treatment （P〈0.01）. Pamidronate increased OPG mRNA levels by primary rat osteoblasts in a dose-dependent fashion （P〈0. 01）. At the most effective dose of pamidronate （10^-6 M for 24 h）, the induction of OPG mRNA was 2. 2-fold. Pamidronate decreased OPG gene expression at a concentration of 10^-5 M, which may be due to the cytotoxic effect. Treatment with pemidronate at a concentration of 10^-6 M inhibited RANKL mRNA levels and increased OPG mRNA expression in a time-dependent manner with a maximum after 24 and 48 h, respectively （P〈0.01）. Conclusion Pamidronate stimulated production of OPG, while inhibited the mRNA levels of RANKL in primary rat osteoblasts, which may, at least in part, be related to the inhibitory effects of bisphosphonates on the osteoelastic bone resorption.