IL-1β和TNF-α对成骨样MG63细胞OPG和RANKL表达的影响

Effects of IL-1β and TNF-α on the expression of OPG and RANKL in osteoblastic MG63 cells

目的体外环境下,探究白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)对成骨样MG63细胞的核因子κB受体活化因子配体(RANKL)和骨保护素(OPG)表达的影响。方法单独或联合使用IL-1β和(或)TNF-α刺激成骨样MG63细胞24 h,分别设为IL-1β干预组、TNF-α干预组、联合干预组(TNF-α及IL-1β联合干预),另设无任何刺激的空白组。Western blot和RT-PCR法检测RANKL和OPG蛋白及mRNA的表达。另用RANKL序列特异性小干扰RNA(siRNA)及非靶序列siRNA,经过Liopfcctamine 2000转染MG63细胞,非靶序列siRNA转染的细胞为阴性对照组,然后使用IL-1β和TNF-α联合刺激24 h,通过Western blot法检测RANKL和OPG蛋白的表达。结果与空白组相比,IL-1β干预组和联合干预组的RANKL和OPG表达量明显提升(P<0.05)。IL-1β及TNF-α联合干预24 h后,RANKL-siRNA转染后的MG63细胞RANKL和OPG蛋白和mRNA表达较非靶序列siRNA转染的阴性对照组显著下调(P<0.05)。结论 IL-1β和TNF-α通过改变RANKL/OPG影响骨代谢,RANKL沉默可以显著降低RANKL蛋白表达,进而影响RANKL/OPG。

Objective To investigate the effect of IL-1β and TNFα on the expression of osteoprotegerin（ OPG）and receptor activator of nuclear factor-κB ligand（ RANKL） in osteblast-like cells MG63. Methods Osteoblast-like cell MG63 were stimulated by IL-1β and TNF-α alone or in combination for 24 hours. IL-1β stimulation group,TNF-α stimulation group and IL-1β and TNF-α stimulation group were established,and blank control group without any stimulation was also established. Real-time PCR was employed to detect the expression of RANKL and OPG mRNA.Western blot was employed to analyze the expression of RANKL and OPG. The effective interference sequence and non-specific small interferenceRNA（ siRNA） were transfected into osteoblast using Lipofectamine 2000. The non-specific siRNA was used as control. After IL-1β and TNF-α intervention for 24 hours,Western blot was employed to analyze the expression of RANKL and OPG. Results After intervention for 24 hours,IL-1β stimulation,and IL-1β and TNF-α combined stimulation significantly up-regulated the OPG mRNA and protein expression than blank control group（ P 〈0. 05）,also the RANKL mRNA and protein expression（ P〈 0. 05）. Compared with non-specific siRNA group,the expression of RANKL and OPG mRNA in MG63 cells with RANKL-siRNA was significantly down-regulated. Conclusion IL-1β and TNFα may have an influence on bone metabolism through regulating RANKL/OPG ratio. RANKL silencing by siRNA could significantly reduce the expression of RANKL and make an impact on RANKL/OPG ratio.