【作者】 朱庆林；

【导师】 吴补领； 费俭； 余擎；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2005， 博士

【摘要】 牙齿的发育矿化是上皮和间充质相互作用的结果,这是一个由众多信号通路参与的复杂过程,这些通路从细胞外的各种信号分子开始,经过表面受体、跨膜信号转导,胞内信号传递等一系列过程,引发特异性基因的表达,导致了特异性的细胞生物学行为,作为体内非常特殊的一种钙化器官,牙齿的发育过程尤其体现了这样一种特异性行为。作为引发钙化的成釉细胞与成牙本质细胞这两种特殊的细胞,二者分泌的基质特异性蛋白是形成这两种矿化组织的关键,成釉细胞所分泌的釉原蛋白是釉质形成过程中含量最丰富的蛋白,它的缺陷可以直接导致遗传性牙釉质发育不全的产生,那么是什么因素导致了这种蛋白在成釉细胞表达的组织特异性呢?通常人们在这方面最为关注的是转录环节的问题。因此,寻找重要的转录调控因子,便成为一个重要的研究内容,Cbfα1是一种成骨分化特异性转录因子,许多矿化相关蛋白如ALP、OC、OPN、BSP等均受其调控。基因敲除的小鼠不仅全身骨发育异常,还出现牙齿发育障碍。Cbfα1的转录因子特性和它在牙齿发育过程中的表达特征提示其可能具有调节釉原蛋白的转录活性的能力。以下进行的各项研究验证了这个设想。 通过对釉原蛋白编码区上游8.5k bp的区域进行检索和对这段序列上是否具有潜在的Cbfα1结合位点进行的分析,发现釉原蛋白的上游序列中的确存在可能与Cbfα1结合的位点。根据最为严谨、也是比较公认的一种核心序列的碱基排列方式—PuACCPuCA,总共有三个位点最为可能,其中两个分别位于转更多还原

【Abstract】 Tooth development and mineralization is regulated by the sequential and reciprocal interactions between the epithelial and mesenchymal tissues ,Which is composed of complex signaling network, including growth factors, their receptors, transmembrane signal transduction, transcription factors and intracellular signal pathway. Theses pathway induce some special gene expression and affect some special biological behavior of these cells. As a special biomineralized organ, tooth development is an important model of these procedure. The extracellular matrix proteins secreted by ameloblast and odontoblast are the key protein for the development of enamel and dentin. Amelogenin is the most abundant extracellular matrix protein in enamel developing. Amelogenin-deficiency can cause amelogenesis imperfecta(AI). What is the reason of the cell specific gene expression? The most important thing we care of is transcriptional regulation. So to find the key trancription factors is very important for us to learn the transcription information. Cbfal is a strong regulator for osteo-specific gene expression, such as ALP, OC, OPN, BSP. The Cbfal-deficient mice also showed abnormal dental development. According to the expression characteristic of Cbfal in tooth development. We soppose that Cbfal can regulate the trancription activity of Amelogenin. To prove the suppose, we did the experiment as followed.Fitst, the 8.5 kbp secquence of 5’flanking of Amelogenin gene was anylized,and some potential Cbfal binding sites were found. According to the most common core binging secquence-PuACCPuCA. 3 binding sites at -5224, -1641 and +92 were found. To analyse all the transcription region, we amplified the overlapped secquence3760bp and 3035bp by PCR from the genomic DNA of mouse C57BL/6J. By restriction endonucleases enzyme digestion and ligation, 5 transcriptional regulation sequences different in length of the 5’flanking of Amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3 -Basic vector.These report genes were transient transfected into CHO, Hela and UMR-106 cells and luciferase assay was performed to analyze the transcription activation of these promoters. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Hela cell can be used as a good model to study the transcriptional regulation of Amelogenin promoter. According to the different activity of different length, it is suggested that there were some potential siliencer located between -1693 and -975, and some potential activator in the region between -532 and -285. The longest promoter report gene was co-transfected into Hela, CHO, and UMR-106 cells with Cbfal to compare the different roles of Cbfal in Hela, CHO, and UMR-106 cells. Cbfal served as a strong transcriptional activator on Amelogenin promoter in Hela cells. In CHO and UMR-106 cells, Cbfal showed weak activation. It proved that this activation is epithelium-specific. To find the binding site of Cbfal in the promoter region, the activitation of Cbfal was systematically tested in Hela cell by co-transfection of Cbfal with 6 different Amelogenin promoter report genes. In this way, we found that activation of Cbfal on single site didn’t upgulate in large-scale . It seemed that the up-regulation is related to cooperation of muti-sites . To confirm the conclusion of bio-information analysis, three report vector deleted or mutated on -5224, -1641 and +92 are constructed. Co-transfected with Cbfal to Hela cells, the three sites showed different activation. -1641 and +92 are concerned with the up-regulated expression. -5224 showed weak activation.To further prove the two site -1641 and +92 were the binding sites of Cbfal.更多还原