体外RNA干扰抑制RANKL表达对成骨细胞功能的影响  

The influence of suppression of RANKL expression by RNA interference on the function of osteoblast in vitro

作　　者：高延征[1] 高坤[1] 于正红[2] 司文腾[1] 

机构地区：[1]河南省人民医院骨科,河南郑州450003 [2]河南省中医学院第二附属医院骨科,河南郑州450002

出　　处：《河南医学研究》2009年第1期1-5,共5页Henan Medical Research

摘　　要：目的:观察用RNA干扰的方法抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator ofNF-κB,RANKL)mRNA表达后,成骨细胞RANKL、OPG蛋白表达和I型胶原表达、细胞增殖能力的时间变化规律。方法:设计4条RANKL序列特异性小干扰RNA(siRNA),用Lipofectamin2000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检测转染后1、2、3、5、7天RANKL、OPG蛋白的表达,同时观察成骨I型胶原表达、细胞增殖能力在相同时间点的变化。结果:对siRNA中有一对可使大鼠成骨细胞的RANKLmRNA水平下降89%。用最佳siRNA转染后1、2、3、5、7天,与空白对照组相比,RANKL蛋白表达率分别为59.1%、39.5%、26.6%、40.0%、57.3%(P<0.05),OPG蛋白表达率较空白对照组降低,但差异不具有显著性(P>0.05)。成骨细胞的I型胶原表达、增殖能力在相同时间点较空白对照组降低,差异也不具有显著性(P>0.05)。结论:靶向siRNA可显著下调成骨细胞RANKL表达,同时对OPG表达及成骨细胞的主要功能无显著影响。Objective： To investigate the change of ligand of receptor activator of NF-КB （RANKL） , Osteoprotegerin（OPG） and the function of osteoblast at different times after suppression of the expression RANKL by RNA interference. Methods ： Four pairs small interference RNA （siRNA） targeting RANKL were designed and transfected into osteoblast using Lipofectamin2000. RANKL mRNA level was determined by real-time quantitative reverse transcriptase polymerase chain reaction（RT-PCR） to screen the most effective siRNA. Western blot was employed to analyze the expression of RANKL and OPG at 1,2,3,5,7 days after transfecting. Proliferation activity and I type collagen expression of osteoblasts were observed at the same time. Results ： The most effective siRNA found out among the 4 candidates. Single dose of this siRNA caused nearly 89% loss of RANKL mRNA. Compared with the control group, RANKL protein in osteoblast decrease to 59.1% ,39.5% ,26.6% ,40.0% ,57.3% （P 〈0.05 ） respectively at 1,2,3,5,7 days after RNAi treatment. The expression rate of OPG protein is lower than the control group at 1 , 2, 3, 5, 7 days after transfection, however no statistical difference existed （P 〉 0.05）. Proliferation activity and I type collagen expression of osteoblasts were similar to the expression profile of OPG （ P 〉 0.05）. Conclusion： These results provided specific siRNA can significantly decrease the expression of RANKL of osteoblast, and the expression of OPG and the major function of osteoblast were not influenced significantly.

关 键 词：核激活因子受体配体 RNA干扰 大鼠 成骨细胞 基因沉默 

分 类 号：R739[医药卫生—肿瘤]