RNA干预法特异性抑制核激活因子受体配体诱导破骨细胞的形成

RNA interference to inhibit osteoclastogenesis induced by receptor activator of nuclear factor-kappa B ligand

目的:使用RNA干预来特异性减少核激活因子受体配体的表达从而抑制破骨细胞的形成。方法:实验于2005-01/2006-10在解放军第四军医大学全军骨科研究所完成。①构建由U6启动子引导产生小干预RNA的质粒载体mU6pro-dsRANKL。②按0.5,1.0,1.5,2.0,2.5,3.0μg的梯度将质粒mU6pro-dsRANKL及对照质粒mU6pro转染大鼠骨髓基质细胞,48h后提取细胞总RNA。③采用Northern blot法检测,以核激活因子受体配体与内参β-肌动蛋白扩增产物的吸光度比值来反映核激活因子受体配体mRNA的表达情况。④再将质粒mU6pro-dsRANKL按0,1.0,2,3μg转染破骨细胞,抗酒石酸染色显微镜下观察。结果:①Northern blot结果:显示转染骨髓基质细胞后细胞中核激活因子受体配体mRNA的表达量随着质粒载体量的增加而减少。②破骨细胞抗酒石酸染色显示:破骨细胞的数量也随着质粒载体量的增加而减少。结论:U6启动子引导产生小干预RNA的质粒载体能有效的减低核激活因子受体配体的表达从而抑制破骨细胞的形成。

AIM： To inhibit osteoclastogenesis by specially decreasing the expression of receptor activator of nuclear factor-kappa B ligand （RANKL） with RNA interference. METHODS： The experiment was conducted in the Research Institute of Orthopedics, Xijing Hospital, Fourth Military Medical University of Chinese PLA between January 2005 and October 2006. （1）mU6pro-dsRANKL, the plasmid vector of short interfering RNA （siRNA） was constructed induced by U6 promoter. （2）The bone marrow stromal cells were transfected with 0.5 μg, 1.0 μg, 1.5μg, 2.0 μg, 2.5 μg and 3.0 μg mU6pro-dsRANKL and the control plasmid mU6pro, respectively. Forty-eight hours later, the total RNA of cells was isolated. （3）Northem blot was used to detect the RANKL mRNA expression through the absorbance ratio of northem blot products of RANKL and β-actin mRNA. （4）Then the osteoclasts were transfected with 0 μg, 1.0 μg, 2 μg, 3 μg mU6pro-dsRANKL, and observed by microscope after tartrate-resistant acid staining. RESULTS： （1）Northem blot results suggested that the level of RANKL mRNA expression in bone marrow stromal cells was decreased with the increase in quantity of mU6pro-dsRANKL. （2）The number of osteoclasts was decreased dramatically as the quantity of mU6pro-dsRANKL increased. CONCLUSION： The plasmid vector of siRNA induced by U6 promoter could effectively reduce the mRNA expressions of RANKL, and finally inhibit osteoclastogenesis.