Runx 2在牙釉质形成中的作用研究

Effect of Runx 2 in developing dental enamel

目的:研究小鼠成釉细胞中Runx 2对釉成熟蛋白(Amelotin,AMTN)、成釉蛋白(Ameloblas-tin,AMBN)、牙成釉细胞相关蛋白(odontogenic ameloblast-asssociated protein,ODAM)基因表达的调控作用,为研究Runx 2在牙釉质形成中的作用奠定基础。方法:通过RT-PCR法从小鼠成釉细胞中克隆得到Runx 2全长基因,测序正确后将其亚克隆至pcDNA3.1-hisA载体中;构建针对小鼠Runx 2的siRNA表达框架,将反应质粒pcdna-3.1-HisA-Runx 2及针对Runx 2的特异性小分子干扰RNA(siRNA)分别转染小鼠成釉细胞,用RT-PCR方法检测Runx 2,AMTN,AMBN以及ODAM的mRNA水平。结果:转染的小分子干扰RNA(siRNA)对Runx 2mRNA的表达有显著性抑制作用,Runx 2基因沉默可显著抑制AMTN和AMBN的mRNA表达水平;Runx 2基因过表达可显著上调AMTN和AMBN的mRNA表达水平。Runx 2基因的表达变化对ODAM的mRNA表达水平则无显著性调控作用。结论:Runx 2通过调控AMTN和AMBN的表达水平,在牙釉质形成过程中发挥作用。

AIM： To investigate the regulating effects of Runx 2 on the expression of Amelotin（AMTN）,Ameloblastin（AMBN） and odontogenic ameloblast-asssociated protein（ODAM） in ameloblasts.METHODS： A full length cDNA of Runx 2 was cloned from mouse ameloblast-lineage cell（ALC） by RT-PCR,and then it was sub-cloned into the pcDNA3.1-HisA plasmid after sequence analysis.One specific dicer siRNA targeted to Runx 2 mRNA was designed and sythesized.The recombinant plasmid pcDNA3.1-HisARunx 2 and the siRNA for Runx 2 mRNA was transfected into ALC respectively.Quantitative real-time PCR was performed to measure the expression of Runx 2,AMTN,AMBN and ODAM gene.RESULTS： The siRNA-Runx 2 elicited a high level of gene silence in ALC.siRNA for Runx 2 effectively inhibited the expression of AMTN and AMBN.Runx 2 overexpression by pcDNA3.1-HisA-Runx 2 plasmid transfection resulted in a significant higher mRNA level of AMTN and AMBN,but exerted no significant effect on the expression of ODAM.CONCLUSION： Runx 2 may play an important role in developing dental enamel by regulating the expression of AMTN and AMBN.