RNA干扰沉默VDR基因对小鼠成骨细胞Dmp1?Cbfa1及BMP-2 mRNA 表达的影响
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南京市卫生局医学科技发展项目(ZKM06045);南京医科大学重点发展项目(06NMU2024)


Effects of vitamin D receptor interference on the mRNA expression of Dmp1,Cbfa1 and BMP-2 in mouse osteoblast mc3t3-E1 cells
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    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VDR mRNA 144-594-852-3 628 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24 h及72 h后收集细胞分别抽提RNA及蛋白-采用RT-PCR 法检测细胞中VDR mRNA 表达水平的改变,Western blot法检测VDR 蛋白表达的变化,从而筛选有效序列-进一步应用SYBR Green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VDR mRNA 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VDR mRNA 852-3 628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-

    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.

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李婵娟,顾海燕,赵德育,刘晓梅,张 翔. RNA干扰沉默VDR基因对小鼠成骨细胞Dmp1?Cbfa1及BMP-2 mRNA 表达的影响[J].南京医科大学学报(自然科学版),2009,29(5):669-673

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  • 收稿日期:2008-12-23
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