【摘要】 目的:利用RNA干扰技术,阻断VDR在小鼠成骨细胞株mc3t3-E1中的表达,观察VDR表达受抑后对小鼠成骨细胞Dmp1、Cbfa1及BMP-2基因mRNA表达的影响。方法:针对小鼠VDRmRNA144、594、852、3628位点设计、合成4对21核苷酸siRNA(siRNA1、siRNA2、siRNA3、siRNA4);在阳离子脂质体介导下转染mc3t3-E1细胞,以空白及非特异性siRNA作为对照,各组于转染24h及72h后收集细胞分别抽提RNA及蛋白。采用RT-PCR法检测细胞中VDRmRNA表达水平的改变,Westernblot法检测VDR蛋白表达的变化,从而筛选有效序列。进一步应用SYBRGreen荧光实时PCR方法定量检测成骨细胞功能基因-Dmp1、Cbfa1及BMP-2基因mRNA表达情况。结果:与空白对照组相比,转染siRNA3、siRNA4的mc3t3-E1细胞VDRmRNA和蛋白表达明显下调(P<0.01),转染siRNA1、2及非特异性siRNA的mc3t3-E1细胞VDR的表达与对照组相比无显著差异(P>0.05)。荧光定量PCR结果显示,转染siRNA3、siRNA4的mc3t3-E1细胞Dmp1、Cbfa1、BMP-2mRNA表达明显下调(P<0.01)。结论:针对小鼠VDRmRNA852、3628位点设计、合成的siRNA可有效抑制小鼠成骨细胞株VDR的转录和表达;VDR表达受抑可下调mc3t3-E1细胞功能基因Dmp1、Cbfa1、BMP-2mRNA的表达;VDR在维持成骨细胞功能中起着一定的作用。更多还原

【Abstract】 Objective:To investigate the inhibition effect of small interfering RNA(siRNA)on the expression of vitamin D receptor(VDR),and observe the effects of down-regulation of VDR on the expression of dentin matrix protein 1(Dmp1),core binding factorα1(Cbfa1)and bone morphogenesis protein 2(BMP-2)mRNA in mouse osteoblast mc3t3-E1cells.Methods:Four pairs of 21 nucleotide VDR siRNAs directed to mouse VDR mRNA 144,594,852 and 3 628 targets were transfected into mc3t3-E1cells with LipofectamineTM 2000,while untreated and transfected scramble siRNA served as the blank control and nonspecific siRNA control separately.Total RNA of the cells were extracted after 24 h of transfection while protein extracted after 72 h.The expression of VDR in mRNA and protein levels were assessed by semiquantitive RT-PCR and Western blot respectively,according to ascertainment of the effective siRNA sequences.The expression of the functional gene of osteoblast,Dmp1,Cbfa1 and BMP-2 mRNA in mc3t3-E1 cells were detected by quantitive SYBR Green real-time PCR.Results:Compared with the blank control group,the expression of VDR mRNA and protein were significantly down-regulated by siRNA directed to 852 and 3628 target of mouse VDR mRNA(P < 0.01),while siRNA directed to 144 and 594 targets showed no effect on the expression of VDR(P > 0.05).According to the results of real-time PCR the expression of Dmp1,Cbfa1 and BMP-2 mRNA were down-regulated in mc3t3-E1 cells transfected with siRNAs directed to 852 and 3628 targets(P < 0.01).Conclusion:VDR siRNA can effectively inhibit the mRNA and protein mRNA expression of VDR.Inhibition of VDR expression down-regulates the mRNA expression of Dmp1,Cbfa1 and BMP-2 in mouse osteoblastic mc3t3-E1 cells.VDR may play some role in the maintenance of osteoblast functions.更多还原