小鼠牙本质涎磷蛋白(DSPP)特异性启动子的克隆和序列分析

目的 :获得小鼠牙本质涎磷蛋白 (DSPP)编码序列上游约 1.6kb的特异性启动子。方法 :按Mac Dougall报道设计一对引物 ,提取小鼠胚胎干细胞基因组DNA作为模板 ,PCR获得大小约为 1.6kb的DNA片段 ,将该片段克隆并进行序列分析。结果 :所得到的片段是DSPP的特异性启动子 ,其序列与文献报道有一定差异 ,但转录因子的结合位点均未发生突变。结论 :成功获得小鼠DSPP的特异性启动子 ,为进一步的研究打下基础。     

Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci

Dentinogenesis imperfecta type II and dentin dysplasia type II are diseases resulting in abnormal dentin formation, which have been mapped to overlapping regions of human chromosome 4q defined by markers D4S2691 and D4S2692 (6.6 cM) and D4S3291 and SPP1 (14.1 cM), respectively. Recently, two of the major non-collagenous proteins of dentin, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP, phosphophoryn) have been shown to be encoded by a single gene, termed dentin sialophosphoprotein (DSPP), which has been mapped to human chromosome 4. The purpose of this study was to perform refined mapping of DSPP related to these disease loci by gene content mapping, as well as to place the DSPP gene on the physical map of human chromosome 4 by sequence tagged site (STS) content mapping. Human genomic DSPP clones were isolated, and gene content mapping performed with specific primers for dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (secreted phosphoprotein 1, SPP1). STS content mapping was then performed with flanking STS markers to these dentin/bone gene loci. Our results demonstrate that the DSPP and DMP1 genes are within a maximum distance of 110 kb. Both DSPP and DMP-1 have been placed on the physical map of human chromosome 4 within the interval defined by markers D4S564 and D4S1292. DSPP is thereby strengthened as a candidate gene for both DGI-II and DD-II.     

DSPP基因启动子不同片段启动活性的对比分析

目的：对比分析牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP）基因启动子不同片段的启动活性。方法：选择小鼠成牙本质细胞样细胞MDPC-23为实验细胞,利用含DSPP基因启动子不同片段的真核表达载体,采用报告基因方法观察DSPP基因启动子不同片段的启动活性。结果：在MDPC-23细胞中,DSPP基因启动子不同片段的真核表达载体均不同程度地表现了启动活性,其启动活性有差异（P〈0.01）。结论：DSPP基因启动子不同片段的启动活性有差异,其活性变化反映位于-95bp～54bp、-410bp～-195bp、-1243bp～-791bp、-1447bp～-1243bp、-3519bp～-2475bp区存在潜在的增强子;-195bp～-95bp、-670bp～-410bp、-2475bp～-1447bp区存在潜在的抑制子。进一步明确了DSPP基因启动子的结构,为今后研究DSPP基因的特异性表达奠定基础。     

牙本质涎磷蛋白的研究进展

牙本质涎磷蛋白及其蛋白剪切产物—牙本质涎蛋白和牙本质磷蛋白是在牙本质发育、矿化中起重要作用的非胶原蛋白.关于牙本质涎磷蛋白的结构、功能、表达以及翻译后修饰等均有大量的研究,使之对牙本质涎磷蛋白的认识不断深入.本文就近年来关于牙本质涎磷蛋白的研究现状作一综述.     

Dentin sialoprotein isoforms: detection and characterization of a high molecular weight dentin sialoprotein

Dentin sialoprotein (DSP) is a glycoprotein accounting for 5–8% of the dentin non-collagenous proteins. The cDNA sequence predicts that rat DSP has 13 potential casein kinase phosphorylation sites and six potential N -linked glycosylation sites. However, its total phosphorylation level, as well as the nature and locations of the carbohydrate moieties, are unknown. Our findings in the present study show that rat DSP has 6.2 phosphates per molecule and that the majority of carbohydrates are attached to the protein through N -linked glycosylations. During our separation of dentin non-collagenous proteins with ion-exchange chromatography, we observed high molecular weight components eluting late in the salt gradient that were recognized by anti-DSP antibodies. We have purified these high molecular weight components using a monoclonal anti-DSP antibody affinity column. Data from amino acid analysis, phosphate level measurements and Edman degradation of tryptic peptides unequivocally proved that the very acidic, high molecular weight components are isoforms of DSP (designated HMW-DSP). Deglycosylation analysis indicates that the slower migration rate of HMW-DSP on SDS-PAGE results from its higher level of carbohydrate modifications.     

Role of the NH2‐terminal fragment of dentin sialophosphoprotein in dentinogenesis

Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH₂‐terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP‐PG)] and a COOH‐terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH₂‐terminal fragment of DSPP (i.e. DSP and DSP‐PG) in dentinogenesis remain unclear. This study focuses on the function of the NH₂‐terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH₂‐terminal fragment of DSPP driven by a 3.6‐kb type I collagen promoter (Col 1a1) were generated and cross‐bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH₂‐terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis.     

小鼠牙胚、软骨组织中牙本质涎磷蛋白基因的克隆

目的：从小鼠牙胚、软骨组织中克隆牙本质涎磷蛋白基因（dentin sialophosphoprotein,DSPP)。方法：采用RT-PCR方法,从小鼠牙胚及软骨组织中克隆DSPP基因片段,将所得片段装入载体pGEM-TEasyVector进行序列测定。结果：从两种组织中均获得719bp的特异性片段,序列分析表明,与已发表的DSPP序列99.7%同源。结论：成功地从小鼠牙胚。软骨中克隆到DSPP基因部分序列。结果提示：除牙齿组织外,DSPP还可在软骨中表达,它可能并非是检测成牙本质细胞的特异性指标。     

Identification of the DSPP mutation in a new kindred and phenotype-genotype correlation

Oral Diseases (2011) 17 , 314–319 Objective: Hereditary dentin defects can be grouped into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia. Tooth enamel is considered normal in patients with hereditary dentin defects, but is easily worn down and fractured due to DSPP mutation‐induced altered dentin properties. The purposes of this study were to identify genetic cause of a family with type II DGI and enamel defects. Materials and methods: We identified a family with type II DGI and a unique form of hypoplastic enamel defect affecting occlusal third of the crown. Family members were recruited for the genetic analysis and DNA was obtained from peripheral whole blood. Results: Mutational analysis revealed a T to A transversion in exon 3 of the DSPP (c.53T>A, p.V18D). Haplotype analysis showed that the same mutation arose separately in two different families having DGI with similar enamel defects, indicating that this phenotype is associated with this specific DSPP mutation. Clinical features suggest that enamel formation was affected in the affected individuals during early amelogenesis, in addition to the dentin defect. Conclusions: We observed that a DSPP gene mutation not only influences dentinogenesis but also affects early stage amelogenesis.     

小鼠牙本质涎磷蛋白5′端非编码区启动子报告基因载体的构建和分析

目的：克隆小鼠牙本质涎磷蛋白(dentin sialophosphopmtein，DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性：方法：PCR、报告基因载体构建、瞬时转染和报告基因检测。结果：PCR获得DSPP启动子的3个不同片段，将它们克隆到萤火虫荧光素酶报告基因载体pG13-Enhancer，构建出3种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

DSP基因编码区序列的多态性研究

目的:分析中国人群中牙本质涎蛋白基因编码区序列的多态性。方法:采用聚合酶链式反应-单链构象多态(PCR-SSCP)分析方法,并结合DNA直接测序方法对牙本质涎蛋白基因编码区的核苷酸序列进行分析。结果:在牙本质涎蛋白基因编码区序列中发现了3个单核苷酸多态(cSNP),其中2个为同义cSNP,编码的氨基酸未变,1个为非同义cSNP,编码的氨基酸分别为天冬氨酸和天冬酰氨。结论:中国人群中牙本质涎蛋白基因编码区序列中存在单核苷酸多态。     

早期牙髓损伤修复中牙本质涎磷蛋白表达的免疫组化研究

目的：衬步探讨牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP)在早期早贿损伤修复中的作用。方法：制备大鼠牙髓损伤模型,分别对损伤后6h,12h,1d,2d,3d,7d及正常大鼠牙髓标本进行HE和免疫组织化学染色,检测各实验组标本中DSPP的表达。结果：正常大鼠牙髓中仅在成牙本质细胞层有DSPP的阳性着色;牙髓损伤后6h,12h,DSPP在成牙本质细胞层的染色明显减弱;损伤后1-3d,梁色较正常牙增强。此时,牙尖及富细胞区的牙髓细胞也有了性着色;损伤后7d,染色与正常对照无明显差异。结论：DSPP在牙髓损伤的早期阶段表达下调;随后在成牙本质细胞及富细胞区牙髓细胞中表达增强;7d时表达趋于正常。提示：它可能在牙髓损伤早期的自身修复中起作用。     

牙本质涎蛋白在氟中毒大鼠牙髓组织和牙本质中的表达和意义

目的:研究氟中毒对大鼠牙髓及牙本质表达DSP的影响。方法:选择20只Wistar大鼠,随机分为对照组(饮用自来水,水氟浓度0.16 mg/L)和给氟组(水氟浓度100 mg/L)。3个月后处死大鼠,利用HE染色、免疫组化染色观察氟中毒对牙本质结构和DSP表达的影响。结果:100 mg/L组牙本质生长线明显加重,出现大量球间牙本质。DSP蛋白在牙本质、成牙本质细胞、牙髓细胞中均有不同程度的表达,在牙本质层内侧的成牙本质细胞和牙髓细胞的染色强度2组间无明显区别(P>0.05)。在给氟组,强烈的DSP染色持续出现在前期牙本质层和矿化的牙本质层(P<0.05),表现为加重的生长线。结论:DSP在氟中毒组牙本质中表达明显增强,推测氟可能影响DSP蛋白的降解,影响牙本质的正常矿化。     

小鼠牙本质涎蛋白成熟肽编码区cDNA克隆和部分序列分析

目的：克隆小鼠牙本质涎蛋白（ＤＳＰ）成熟肽编码区基因。方法：用异硫氰酸胍一步法从昆明新生小鼠磨牙牙胚组织中抽提总ＲＮＡ,用Ｏｌｉｇｏ（ｄｔ）作引物逆转录合成牙胚ｃＤＮＡ,然后利用ＰＣＲ方法,从ｃＤＮＡ中扩增出小鼠ＤＳＰ成熟区的基因片段（约１．４ｋｂｐ）,将所得基因片段插入ｐＢＳ质粒载体,转化到大肠杆菌ＸＬ１－Ｂｌｕｅ后挑选阳性克隆,提取重组质粒ＤＮＡ,通过限制性酶切和核苷酸序列分析鉴定阳性克隆。结果：重组质粒ｐＢＳ－ＤＳＰ的酶切图谱和部分序列分析结果与国外文献报道一致。结论：克隆到小鼠ＤＳＰ成熟肽编码区基因,正在进行该基因的表达和活性鉴定。     

牙本质磷蛋白和成釉蛋白在组织工程牙胚的表达模式研究

目的：采用大鼠牙胚细胞与胶原蛋白构建组织工程牙齿,通过免疫荧光组织化学分析牙齿特异性蛋白在组织工程牙齿的表达模式。方法：采用出生后4dSD仔鼠磨牙牙胚细胞,培养后将牙胚细胞与胶原蛋白溶液混合构建组织工程牙胚,2W后取材,HE染色观察移植物中的成牙能力,免疫荧光组织化学染色观察牙本质磷蛋白DSP和成釉蛋白AM的表达模式。结果：组织工程牙胚移植后2W可在肾被膜下形成乳白色矿化组织,HE染色可见典型的牙髓牙本质复合体样组织和釉质样结构,免疫荧光组织化学染色观察可见成釉蛋白AM阳性的釉质样结构和牙本质磷蛋白DSP阳性的牙乳头样组织,与正常牙齿组织表达模式一致。结论：采用牙胚细胞可与胶原蛋白进行重新排列形成组织工程牙胚,本实验从组织和蛋白分析上证实了其具有较强的成牙能力,为以后的临床应用提供了理论基础。     

小鼠牙本质涎磷蛋白启动子报告基因载体的构建和启动子活性分析

目的：克隆小鼠牙本质涎磷蛋自（dentin sialophosphopmtein,DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性。方法：细胞基因组提取，PCR，瞬时转染和报告基因检测。结果：从MDPC-23细胞基因组中克隆出长为1.5kbp的DSPP启动子，将启动子酶切成不同的片断，克隆到虫工业基础光素酶报告基因载体pC1.3-Enhancer,构建出4种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

人牙本质涎蛋白在人牙胚发育过程中的表达和意义

目的：探讨人牙本质涎蛋白(human dentin sialoprotein，hDSP)在人牙胚发育过程中的表达和意义。方法：用免疫组化方法检测人牙本质涎蛋白在人牙胚不同发育阶段中的表达。结果：hDSP在蕾状期、帽状期釉上皮以及钟状早期内釉上皮有弱阳性表达，钟状中期正在分泌基质的成牙本质细胞、牙本质小管有强阳性表达，钟状晚期，牙本质小管仍有强阳性表达，而成牙本质细胞转为弱阳性表达。前成釉细胞、成釉细胞有一过性表达。前期牙本质始终无阳性表达。牙胚周围骨组织、软骨组织和口腔软组织无阳性表达。结论：提示hDSP可能参与了牙本质的形成。另外前期牙本质阴性表达，表明hDSP蛋白由成牙本质细胞分泌后，可能通过成牙本质细胞突起穿过前期牙本质分泌至矿化前沿，参与牙本质的形成。     

正畸牙根吸收与龈沟液中牙本质涎磷蛋白和牙本质涎蛋白相关性的实验研究

目的 探讨大鼠龈沟液中牙本质涎磷蛋白(DSPP)和牙本质涎蛋白(DSP)的表达与实验性牙移动所致牙根吸收的关系.方法 36只健康Wistardd大鼠随机分成3组:对照组、轻力组、重力组.以上颌切牙为支抗,轻力组和重力组分别以0.392、0.98 N力拉右侧上颌第一磨牙向近中移动.加力7 d后,提取龈沟液,制备实验牙及其...