靶向RANKL基因的siRNA对成骨细胞促破骨细胞生成作用的影响

The Effect of siRNA Targeting RANKL on Osteoclastogenesis Induced by Osteoblast in Vitro

目的利用RNA干扰(RNAi)技术抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator of NF-κB,RANKL)的表达,观察成骨细胞RANKL/OPG比值的变化,探讨成骨细胞与破骨细胞生成的相关性。方法合成4条RANKL序列特异性小干扰RNA(siRNA),用Liopfectamin2000转染成骨细胞,采用荧光实时定量聚合酶链反应(PCR)检测RANKL的表达,筛选出最有效的干扰序列,并用Western blot技术检测成骨细胞的RANKL、OPG的表达,观察RANKL/OPG比例的变化,采用成骨细胞破骨细胞共培养技术探讨转染后的成骨细胞对破骨细胞生成的影响。结果合成的4对siRNA中有一对可使大鼠成骨细胞的RANKL mRNA水平下降89%,蛋白表达下降76%;同时OPG的蛋白表达无明显变化,RANKL/OPG比例明显下调,破骨细胞的生成明显受到抑制。结论RNAi沉默RANKL基因表达可显著下调成骨细胞RANKL/OPG的比值,抑制破骨细胞的生成。

Objective To observe the change of the ratio of ligand of receptor activator of NF-κB（RANKL） and Osteoprotegerin （OPG） in responds to silencing of the expression RANKL with RNA interference（ RNAi ）, and explore the relationship between osteoblast and osteoclastogenesis. Methods Four synthesized small interference RNA （siRNA ） were transfected into osteoblast using Lipofectamin2000.RANKL mRNA level was determined by real-time quantitative reverse transcriptase polymerase chain reaction（ RT-PCR）. Western blot was employed to analyze the expression of RANKL and OPG,then the change of ratio of RANKL and OPG was observed. The tmnsfected cells and primary rat bone marrow cells were cocultured to explore the osteoclastogenesis induced by osteoblast. Results The most effective sequence was found out among the 4 candidates. Single dose of this siRNA caused nearly 89% loss of RNAKL mRNA and 76% loss of RANKL protein in osteoblast, although no significant change was found for OPG.The ratio of RANKL/OPG and the potential of the osteoblasts were significantly reduced. Conclusion These results provided RANKL was vulnerable to RNA interference and RANKL silencing by RNAi could significantly reduce the the ratio of RANKL/OPG and inhibit the formation of osteoclast.