摘要
目的:观察Smad3在转化生长因子β1(TGF-β1)调控人牙本质基质蛋白1(DMP1)转录表达中的作用并对DMP1基因启动子上的结合位点进行初步定位。方法:将Smad3瞬时转染至具有矿化活性的人牙髓干细胞(HDPSC)中,观察Smad3蛋白表达和转位情况,并检测在有无TGF-β1的刺激下细胞中pGL3-P-193～+86和pGL3-P-505～+86启动子活性的变化。pGL3-P-505～+86与Smad3共转染至细胞中,10ng/ml TGF-β1刺激2h后,提取细胞核蛋白,EMSA法检测DMP1基因启动子-505～-193bp区存在的Smad3结合位点。结果:HDPSC中瞬时转染Smad3后,其主要表达于细胞胞浆中,随着TGF-β1刺激不同时间后,Smad3在细胞中的表达出现从胞浆到胞核的转位。Smad3可明显加强TGF-β1下调pGL3-P-193～+86和pGL3-P-505～+86启动子活性的作用,在DMP1基因启动子-505～-193bp区至少有一个Smad3结合区域为-209～-201bp区的GTCTAGTCA序列。结论:Smad3是TGF-β1信号通路的下游作用分子,可介导TGF-β1下调DMP1基因转录过程,DMP1基因启动子-209～-201bp区序列为Smad3结合区域。
Objective：The roles of Smad3 during the regulation of TGF-β1 on DMP1 gene expression in induced human dental pulp stem cells（HDPSC） were examined,and the binding sites of Smad3 in DMP1 promoter sequence was located. Method：The expression conditions of smad3 were examined after transient transfection of Smad3 into the cells,and the transcription activities of pGL3-P-193～＋86 and pGL3-P-505～＋86 were detected after treated with TGF-β1. The potential Smad3 binding sites GTCTAGTCA sequence were preliminarily located by the method of EMSA. Result： The results showed that Smad3 was mostly detected in the cytoplasm of the cells and the transposition of Smad3 was found from the cytoplasm to the nuclei after treated with TGF-β1, which indicated Smad3 participated in the signal pathway in HDPSC. And the transcription activities of pGL3-P-505-＋86 were decreased significantly. EMSA results showed wild oligonucleotides could form protein-DNA complexes which confirmed by the competition tests while the mutant ones could not. In the supershift tests, the more hysteretic staining bands were displayed in the gel which meant that the formation of slower migrating pmtein-DNA complexes. Conclusion： Smad3 was one important downstream molecule of TGF-β1 signal pathway,and the region of -209^-201 bp of DMP1 promoter was Smad3 binding sites.
出处
《临床口腔医学杂志》
2009年第9期524-527,共4页
Journal of Clinical Stomatology
基金
陕西省自然科学基础研究计划项目(2005C221)
