【作者】 高杰；

【导师】 吴补领； 何文喜；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2005， 博士

【摘要】 牙齿的发生源于上皮-间充质细胞的相互作用和相互诱导,从而激发成牙本质细胞和成釉细胞的分化、成熟、合成和分泌细胞外基质,然后羟基磷灰石晶体沉淀,矿化开始。牙本质形成和矿化是牙齿发育和牙齿损伤修复过程中的重要环节。目前研究证实,成牙本质细胞表达和分泌的牙本质非胶原蛋白,特别是牙本质涎蛋白(dentin sialoprotein,DSP)和牙本质磷蛋白(dentin phosphoprotein,DPP)被认为是牙本质特异性蛋白,在牙本质生物矿化、维持矿化组织稳定中发挥着关键的调控作用。这两种蛋白是由同一种蛋白—牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)基因的表达产物裂解而成,而且DSPP基因也是遗传性牙本质发育不全Ⅱ型和Ⅲ型的致病基因,表明DSPP基因的正确表达调控与牙本质形成和矿化密切相关。随着对DSPP基因和蛋白功能研究的进一步深入,DSPP基因的表达调控已经成为目前国内外研究的焦点。 研究表明,DSPP基因的表达调控是一个多因素共同参与的、复杂的过程。其中,TGF-β1作为一种重要的生长因子,在成牙本质细胞分化、牙本质基质形成和矿化过程中均发挥着关键的调控作用,并且可以调控DSPP的表达。现在认为,Smad3是TGF-β1在细胞内的特异性信号分子,主要功能是将TGF-β1信号从胞浆转位至胞核内,调控目的基因的转录。但是Smad3是否能够将TGF-β1信号转位,从而调控DSPP基因的转录与表达,目前尚未见到相关报道。根据Smad3与其它基因的结合元件SBE的序列CAGAC(C/A),或者是其互补序列(T/G)GTCTG,推测其在DSPP启动子上也应该有相应的结合元件。现在已证实,转录因子Cbfal参与了DSPP的转录调控过程,但是Smad3能否和Cbfal协同作用,调控DSPP的表达,也未见到相关报道。因此研究Smad3对DSPP基因表达调控的更多还原

【Abstract】 Tooth development begins because of the reaction and induction between epithelia and mesenchymal cells. This reaction induce odontoblast and enamaloblast to differente and maturate. These cells synthesize and secrete dentin extracellular matrix (ECM). Following this, hydroxyapatite crystal deposits in ECM and mineralizese. Dentin formation and mineralization both are important procedures in tooth development and repair. Many studies show that ECM acts as a key role in these procedures. In ECM, there are two dentin special proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). They regulate the procedure of dentin mineralization and help to preserve dentin stably. These two proteins are cleavage products expressed from a single transcription coded by dentin sialophosphoprotein (DSPP), and DSPP gene is a candidate gene for Dentinogenesis imperfecta Ⅱ /Ⅲ (DGⅠ-Ⅱ, DGⅠ-Ⅲ). So correct expression of DSPP is important during the dentin formation and mineralization.Research has shown the regulation of the expression of DSPP is a complex process influenced by many factors. Among these factors, TGF-β 1 as an important growth factor has been proved to be involved in the regulation of odontoblast differentiation, dentin formation and the expression of the DSPP. Up to date, Smad3 has been considered as specific signalingtranscription factor? Its main function is transporting TGF-β 1 mRNA from cell plasma to cell nucleolus and regulating the transcription and expression of target gene. However, there is no research to show Smad3 is related to the transcription and expression of DSPP gene through the transduction of TGF-β 1 mRNA. According to the SBE of Smad3 [CAGAC(C/A)] or its complementary sequence [GTCTG(T/G)], it is reasonable to consider related SBE also exists in the promoter of DSPP. It has been proved that transduction factor Cbfal is involved in the regulation of the transduction and expression of DSPP, but there is no report on co-effect of Smad3 and Cbfal on the regulation of the expression of DSPP.So, it is helpful to understand the differentiation of odontoblast, the formation of the dentin and the molecules mechanism of the mineralization through the research of regulation of Smad3 to the DSPP. It will provide a new method and idea to prevent caries, pulpitis , DGⅠ-Ⅱ and DGⅠ-Ⅲ.This study used odontoblast line, MDPC-23, to construct DSPP promoter report gene vectors in order to evaluate how Smad3 regulates expression of DSPP, and the role of Smad signaling in odontoblast differentiation and ECM biosynthesis regulated by TGF-β1. Then EMSA was used to study the binding site of Smad3 on DSPP promoter to explore the molecular mechanism of TGF-β1 action during odontoblast differentiation and ECM biosynthesis.The present study consists of three parts:Part one: Function of DSPP promoter Pdspp2.6In this part, we cloned a DSPP promoter from mouse genomic DNA through PCR method. This promoter segment is about 2.6kbp (-2525~+54bp). The amplified 2.6kbp segments were cloned into luciferase report gene vector pGL3 LUC-Enhancer. The orientation of this insert was verified by DNA sequencing and cutting with two different restrict enzymes.Then the pGL3LUC-Pdspp2.6 vector was transfect into MDPC-23 cell. After examination, the results demonstrated that pGL3LUC-Pdspp2.6 had low luciferase activity. When TGF-β1 was used to investigate the effect on DSPP,the result indicated that the DSPP promoter vector was regulated negatively by TGF-β1 in odontoblasts. With the Smad3 existing and over expression, TGF-β 1 had more negative regulation effect on DSPP.Following the study above, we continued the research on the coordination effect of Smad3 and Cbfal on pGL3LUC-Pdspp2.6 activity. Cbfal ’s subtype- Osf2 and PEBP2αA -was used. The results demonstrated that Cbfal could inhibit pGL3LUC-Pdspp2.6 activity in MDPC-23. However, TGF-β1 can inhibit function of Cbfal. That is to say when Cbfal was affect by TGF-β1, pGL3LUC-Pdspp2.6 activity is higher than that without TGF-β1. This result leads to the conclusion indirectly that Cbfal can inhibit expression of DSPP. With Smad3 (wild type) used with Cbfal on pGL3LUC-Pdspp2.6, the results showed much higher inhibition than their single effect. When Osf2, subtype of cbfal, coordinated with Smad3△C, mutant type of Smad3, the results showed no change. But when PEBP2αA, another subtype of Cbfal coordinated with Smad3AC, TGF-β1 can regulate pGL3LUC-Pdspp2.6 activity. These results indicate that PEBP2αA and Osf2 have different function in the transcriptional regulation of DSPP.Part two: Regulation function of Smad3 on DSPP promoter segments Following part one research, in order to clarify the binding site of Smad3 on DSPP promoter, this part continued to construct pGL3LUC-Pdspp vector. First, three DSPP promoter segments, 235bp、 249bp and 464bp, were amplified by PCR. Then they were cloned into the pMD-18T vector by T4 ligase. The orientation of these inserts was verified. Second, the inserted fragments were recloned into pGL3 luciferase(LUC) enhanced expression vector. Then three luciferase report gene vector were constructed, pGL3LUC-Pdspp-410~-176bp, pGL3LUC-Pdspp-195~+54bp and pGL3LUC-Pdspp-410~+54bp. Orientation of all inserts respect to the pGL3LUC vector was verified by restriction enzymes digestion. Then these three vectors were transfected into MDPC-23 cell individually. The results demonstrate that pGL3LUC-Pdspp-410~-176bp and pGL3LUC-Pdspp-410~+54bp have higher promoting activity. As to the pGL3LUC-Pdspp-195~+54bp, although hasbasic promoter, it had low promoting activity. From these results we can conclude that there is one inhibit element at least in DSPP promoter locating in -195~+54bp region.To evaluate the effect of TGF-β1/Smad3 signal on the transcriptional regulation of DSPP, 10ng/ml TGF-β1 was used to evaluate luciferase activity of the three segments mentioned above. The results show that pGL3LUC-Pdspp-410~-176bp and pGL3LUC-Pdspp-410~+54bp can be regulated negatively more significantly by TGF-β1 than pGL3LUC-Pdspp-195 ~+54bp. From these results we can conclude that in the region -410~-176bp of DSPP promoter, binding site of TGF-β1’s signal factor exists.Then wild type Smad3 and mutant type Smad3△C were coordinated with three promoter segments individually. The results show that Smad3 could inhibit promoting activity of pGL3LUC-Pdspp-410~-176bp and pGL3LUC- Pdspp -410~+ 54bp, but it could not inhibit the promoting activity of pGL3LUC- Pdspp -195~+54bp. This result indicates again there is one binding site of Smad3 at least locating in the region -410~-176bp of DSPP. Evidence collected from the experiment past indicates that basic promoter of DSPP is in the region of -95~+54bp, but through our experiment, we can find the -410~-176bp segment also has promoting activity, which indicates DSPP can start its transcriptional progress without basic promoting region.Part three: experimental localization of binding site of Smad3 to DSPP promoterFrom two parts discussed above, we knew the relationship between TGF-β1 and Smad3 during their regulation to DSPP and discovered the binding region of Smad3 exsisting in DSPP promoter.In this part, we first constructed the pMD-18T-Smad3 vector, then recloned Smad3 into the green fluorescent protein (pEGFP-Nl) expression vector. The orientation of insert was verified by cutting with two different restrict enzymes, and the new clone was named pEGFP-Smad3. Then, pEGFP-Smad3 expression vector was transfected into MDPC-23 cells. By the method of immunofluorescence, we discovered green fluorescence in the cell更多还原