【作者】 郭婷；

【导师】 肖明振； 余擎； 赵守亮；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2005， 博士

【摘要】 牙齿是特殊的矿化器官,由釉质、牙本质、牙骨质和牙髓组成,其中牙本质构成牙齿的主体。牙本质起源于牙源性上皮和外胚间充质的相互作用,进而诱导成牙本质细胞分化成熟,合成和分泌细胞外有机基质,然后羟基磷灰石晶体沉积,矿化开始形成。在矿化过程中,细胞外基质起着非常重要的作用。其中最关键的牙本质涎蛋白(dentin sialoprotein,DSP)和牙本质磷蛋白(dentin phosphoprotein,DPP)是目前普遍公认的牙本质特异性蛋白,它们来源于同一个编码基因——牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)基因。长期以来,国内外学者对DSPP基因及其编码蛋白做了大量的研究,包括基因结构、染色体定位、蛋白结构和功能分析等,然而关于DSPP基因转录调控方面的研究报道甚少。 核心结合因子α1(core binding factor α1,cbfα1)是一种成骨细胞特异性转录因子,可以诱导成骨前体细胞合成和分泌ALP、OC、OPN、BSP等矿化相关蛋白,并向成骨细胞分化,因此在成骨分化过程中发挥着至关重要的作用。许多矿化相关蛋白基因的启动子上都已发现有cbfα1的结合位点,如Ⅰ型胶原、OC、OPN、BSP、ALP、金属蛋白酶13等。国内外学者的研究证实cbfα1在牙齿发育过程中存在着时空表达的特异性,其调控的下游蛋白包括多种牙本质基质中重要的矿化组织特异性蛋白。近来的研究又发现由成釉细胞分泌的另外一种牙齿特异性蛋白——成釉蛋白的启动子上也存在2个cbfα1的结合位点,那么cbfα1在DSPP基因的更多还原

【Abstract】 Tooth is a special mineralized organ composed of enamel, dentin, cementum and pulp. Dentin forms the main part of tooth. During dentin formation, instructive epithelial-mesenchymal interactions lead to the cytodifferentiation of ectomesenchymal cells into odontoblasts that produce dentin extracellular matrix (ECM). Previous studies have identified that dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) which are dentin-specific proteins play a very important role during the process of cytodifferentiation and hydroxyapatite crystal multiplication. They are cleavage products of a primary transcript encoded by a single gene termed dentin sialophosphoprotein (DSPP) gene. Many studies have been made about DSPP gene in its structure of gene and protein, mapping, function, and so on. But little is known about the molecular mechanisms of its transcriptional regulation.Core binding factor α1 (cbfα1) is a kind of transcription factor, which plays a key role in osteoblast recruitment and differentiation. It can induce the pro-osteoblasts or non-osteoblasts to synthesize and secrete some mineralization related proteins such as ALP, OC, OPN, BSP, and finally, to differentiate into osteoblasts. Cbfα1 binding site is present in transcriptionalenhancer of many mineralization related gene, such as type I collage, OC, OPN, BSP, ALP, matrix metalloproteinase-13, and so on. Researchers found recently that cbfal is temporally and spatially regulated during dental development. The proteins regulated by cbfal are also very important in dentin mineralization. There are two cbfal binding sites on the mouse ameloblastin promoter.In our study, we investigated the role of cbfal in biomineralization by studying the transcriptional regular function of cbfal on DSPP gene by PCR, gene reconstruction, cell culture in vitro, gene transfection, report gene, electrophoretic mobility shift assays (EMSA), and so on.The present study consists of three parts:1. Clone and construction of the eukaryotic expression vector including the mouse dentin sialophosprotein promoterGenomic DNA was got from mouse blood. The desired DNA segments were obtained by PCR. No.l segment is between nucleotides (nt) -4496bp and -3499bp, No.2 between nt -3519bp and -2515bp, and No.3 between nt -657bp and 17bp. Then the segments were inserted into T-vector to be sequenced. DNA segments of the right clones were cut with restriction enzymes, and subcloned into pGL3-promotor and pGL3-enhancer vector which belong to one kind of report gene of eukaryotic expression vector. The isolated plasmids were identified by digestion with the restriction enzyme. Then, we identified the base between nucleotides (nt) -4496bp and -2515bp. With our partner’s work, we have got the eukaryotic expression vectors of different segments of mouse DSPP promoter as long as 4.4kb.2. The research of transcriptional regulation of cbfal on mousedentin sialophosphoproteinBy transient transfection, immunofluorescence, western blot, and so on, we found that cbfal expression was significantly high 48h after transient transfection. Then we studied the relationship between cbfal and DSPP promoter in different cells. The results showed that cbfal could regulate the transcription of DSPP, and it was more efficient in MDPC-23 cells. After co-transfected pcDNA3-cbfol and report vector of different segments ofmouse DSPP promoter with pRL-TK vector into MDPC-23 cells using the Lipofectamine?2000, the cells were measured for luciferase activity using the dual luciferase reporter assay system. We found that all of the segments of mouse DSPP promoter include the segments of nt -4496bp to -3499bp and nt -3519bp to -2515bp, could drive the expression of the report gene, suggested all the segments had promoter activity. Potential transcriptional enhancers exist in regions between nt -410 and -195, nt -1243 and -791, nt -1447 and -1243, nt -3519 and -2475 base pairs, whereas suppressor elements are located in regions between nt -195 and -95, nt -670 and -410, and nt -2475 to -1447 base pairs. There might have novel potential binding sites for cbfal in the region of nt -4496 to -3499, nt -3519 to -2515.3. The identify of cbfal binding sites on DSPP promoterIn order to identify the binding sites, we designed and synthesized wild and mutant type oligonucleotides for the special region and control segments. For the EMSA, the double-stranded oligonucleotides were labeled with [7-32P]ATP and purified on a 15% polyacrylamide gel. Antibody supershift experiments were performed with specific antibodies to cbfal. The results showed that the wild-type oligonucleotides could form protein-DNA complex, whereas the mutant ones could not. This was confirmed by the competition tests. To further confirm that the sites were cbfal binding site, super-shift experiments were performed using the anti-cbfal antibody. The addition of the antibody led to the formation of slower migrating protein-DNA complexes. This result suggested that there were binding sites for cbfal on DSPP promoter.To test our result, we did some other works in cellular situation. Mutant segments obtained by PCR were inserted into T-vector to be sequenced, and then were subcloned into pGL3-promotor vector. After identification by digestion with the restriction enzyme, the isolated plasmids were co-transfected with pcDNA3-cbfal and pRL-TK vector into MDPC-23 cells. Then the cells were measured for luciferase activity. We found that there were no difference between mutant segments and empty vectors in driving the expression of the report gene, and cbfal couldn’t change the expression level.更多还原