CAJ | 학술논문

目的：：研究Smad3对Amelotin启动子转录活性的影响。方法：用基因克隆技术获得含有不同长度Amelotin启动子片段的荧光素酶报告基因载体,然后将其与Smad3瞬时转染小鼠成釉细胞,并用双荧光素酶报告基因检测其活性；生物学信息软件对人和小鼠的Amelotin基因启动子序列的同源性进行BLAST分析；凝胶迁移实验( EMSA)观察Smad3和Amelotin启动子特异性结合位点之间的相互作用；基因定点突变构建Smad3结合位点突变型Amelotin启动子,并用双荧光素酶报告基因检测系统分析Smad3对野生型和突变型Amelotin启动子转录活性的影响。结果：Smad3转染后, Amelotin启动子(-160~+196)转录活性明显增强(P<0.05)；将人与小鼠的Amelotin启动子序列(-160~+196)进行Blast序列比对,发现两序列在(-160~-1)具有很大的同源性；EMSA结果显示,Smad3与Amelotin启动子(-160~+196)的GTCTG有相互作用；将Amelotin启动子上Smad3结合位点特征性序列“GTCTG”突变为“CCCTG”后,Smad3对突变型Amelotin基因启动子的转录活性无显著影响(P>0.05)。结论：转录因子Smad3可通过与Amelotin启动子特定序列结合而调控Amelotin的基因表达,从而在釉质发育过程中发挥重要作用。
목적：：연구Smad3대Amelotin계동자전록활성적영향。방법：용기인극륭기술획득함유불동장도Amelotin계동자편단적형광소매보고기인재체,연후장기여Smad3순시전염소서성유세포,병용쌍형광소매보고기인검측기활성；생물학신식연건대인화소서적Amelotin기인계동자서렬적동원성진행BLAST분석；응효천이실험( EMSA)관찰Smad3화Amelotin계동자특이성결합위점지간적상호작용；기인정점돌변구건Smad3결합위점돌변형Amelotin계동자,병용쌍형광소매보고기인검측계통분석Smad3대야생형화돌변형Amelotin계동자전록활성적영향。결과：Smad3전염후, Amelotin계동자(-160~+196)전록활성명현증강(P<0.05)；장인여소서적Amelotin계동자서렬(-160~+196)진행Blast서렬비대,발현량서렬재(-160~-1)구유흔대적동원성；EMSA결과현시,Smad3여Amelotin계동자(-160~+196)적GTCTG유상호작용；장Amelotin계동자상Smad3결합위점특정성서렬“GTCTG”돌변위“CCCTG”후,Smad3대돌변형Amelotin기인계동자적전록활성무현저영향(P>0.05)。결론：전록인자Smad3가통과여Amelotin계동자특정서렬결합이조공Amelotin적기인표체,종이재유질발육과정중발휘중요작용。
AIM:To study the regulatory effects of Smad3 on the promoter activity of Amelotin gene in amelo-blasts. METHODS:Luciferase reporter gene vectors containing different lengths of Amelotin promoters were constructed by gene cloning and then the constructs were co-transfected into ameloblasts with Smad3 respectively. Dual luciferase a-nalysis was used to observe the effects of Smad3 on the transcriptional activity of Amelotin gene promoter in ameloblasts. On the basis of above results, the new functional promoter region was found. Electrophoretic Mobility Shift Assay( EM-SA) was carried out to identify the interaction between Smad3 and Smad3 binding site on the Amelotin promoter. The Smad3 binding site on Amelotin promoter were mutated by site-specific mutagenesis and the effect of Smad3 on the tran-scriptional activity of the wild or mutant type Amelotin promoter was observed by Dual luciferase analysis. RESULTS:Transfection of Smad3 increased the Amelotin gene promotor (-160~ +196) transcriptional activity(P<0. 05). Blas-ting analysis showed that promoter sequence(-160~ -1)of Amelotin gene between human and mouse was homologous. EMSA identified the interaction between Smad3 and Smad3 binding site on Amelotin promoter. Mutation of Smad3 bind-ing site did not change the transcriptional activity of Amelotin promoter(P>0. 05). CONCLUSION: Smad3 enhances the transcriptional activity of Amelotin promoter by interacting with Smad3 binding site on Amelotin promoter.