Cbfα1在人牙本质基质蛋白1基因启动子结合位点的初步定位

The location of binding site of core binding factor α1 on human dentin matrix protein 1 gene promoter

目的对核心结合因子α1(core binding factorα1,Cbfα1)在人牙本质基质蛋白1(dentin matrix protein1,DMP1)基因启动子上结合位点进行初步定位。方法 DMP1基因启动子-505^+86 bp区存在成牙本质细胞分化重要的转录调控因子Cbfα1的作用位点,为验证它对DMP1是否具有直接的转录调控作用,将计算机分析结果Cbfα1结合位点(TTGGGAACCACAAGA,-199^-185 bp)进行体外定点基因突变,双酶切和DNA测序鉴定突变效果,突变后的质粒pGL3-P’-505^+86和PCMV-Osf2共转染至体外培养的人牙髓干细胞(human dental pulp stem cells,HDPSCs)中,报告基因检测系统检测虫萤光素酶活性的改变,单纯转染pGL3-P-505^+86组作为空白对照。结果与PCMV-Osf2共转染后,突变pGL3-P’-505^+86共转染组在HDPSCs中的荧光素酶活性较未突变pGL3-P-505^+86共转染组明显下降(P<0.05),突变pGL3-P’-505^+86共转染组较单纯转染pGL3-P-505^+86组活性稍有升高,但无统计学意义(P>0.05)。结论 Cbfα1对DMP1基因转录具有直接的调控作用,DMP1启动子-199^-185 bp区是Cbfα1的结合位点之一。

Objective The location of Cbfc^l binding sites on human DMP1 gene promoter in human dental pulp stem cells is determined. Methods The site-directed mutagenesis of Cbfαl binding sites in the DMP1 promoter DNA sequence （TTGGGAACCACAAGA,-199--185 bp） was undertaken in order to verify their direct controlling roles on the transcriptional expression of DMP1. The mutation results were identified by restriction enzymes cuttings and DNA sequencing, and the luciferase activities were detected after the mutant plasmids were transfeeted into the HDPSC. Results The luciferase activities of mutant co-confection group were obviously decreased than those of un-mutant co- confection group after eo-confeeted with PCMV-Osf2 into HDPSCs （P〈0.05）, and activities of mutant co-confection group were slightly increased than those of un-mutant solo-confection group （P〉0.05）. Conclusion The results confirmed that Cbfal could regulate the transcription expression of human DMP1 gene directly, the fragment from nt -199 to -185 bp on the promoter of DMP1 gene could be one of the binding sites of Cbfα1.