过量氟对大鼠切牙牙本质涎蛋白mRNA表达的影响

目的研究过量氟对大鼠切牙发育过程中牙本质涎蛋白(dentin sialoprotein,DSP)mRNA表达的影响。方法选择6只Wistar大鼠,随机分成两组:对照组常规饲料喂养,自由饮用蒸馏水;实验组常规饲料喂养,自由饮用氟离子浓度为100 mg/L的氟化水,建立大鼠氟斑牙模型。饲养8周处死动物,提取大鼠切牙组织总RNA,逆转录为cDNA,利用实时荧光定量聚合酶链反应技术观察过量氟对大鼠切牙中DSP mRNA表达的影响。结果实时荧光定量聚合酶链反应结果显示DSP mRNA在实验组表达水平明显高于对照组表达水平(t=2.132,P<0.01)。结论过量氟可能通过增强DSP mRNA的表达,影响牙本质的发育矿化。     

牙本质涎蛋白在牙齿发育、牙本质疾病中的表达和作用

牙本质基质是由胶原和非胶原蛋白组成,其中非胶原蛋白又包含了一组牙本质特异性蛋白,它们在牙胚发育,矿化过程中起重要作用.该文就牙本质特异性蛋白中的牙本质涎蛋白在牙齿发育和牙本质疾病中的表达和作用作一综述.     

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。     

bFGF在氟中毒大鼠切牙牙髓中的表达和意义

目的研究过量氟对大鼠牙髓细胞表达bFGF的影响。方法选择20只Wistar大鼠,随机分为2组I组(对照组)和II组(实验组)。8周后处死动物,利用磨片、HE染色、免疫组化染色技术观察过量氟对大鼠切牙形态及bFGF表达的影响。结果实验组大鼠切牙釉质、牙本质生长线明显,牙本质可见钙质小球,牙髓细胞及髓腔内侧牙本质bFGF表达明显弱于对照组,差异有显著性(P<0.01)。结论过量氟可抑制牙髓细胞表达bFGF,影响牙体硬组织的结构。     

人牙本质涎蛋白对狗牙髓损伤修复影响的实验研究

目的 探讨人牙本质涎蛋白对狗牙髓损伤修复的影响。方法 实验共分3组，实验组(hDSP组)；对照l组(无hDSP组)；对照2组(PBS组)；选择10Kg、6～12月龄的杂种狗3只，上、下尖牙、上、下磨牙和下前磨牙为实验牙，每只狗选10个牙，随机分配实验组和对照组，实验组共18个牙，对照l组和2组各6个牙。于所选牙齿颈部，用l／2球钻在灭菌生理盐水滴注下穿通髓腔、露髓处置上述实验组和对照组胶原膜，光固化封闭窝洞。2、4、8周处死动物，经4％多聚甲醛灌注后，分离实验牙，再固定48h后，脱钙3～4周，脱水，石蜡包埋，制备5～8μm厚连续切片，HE染色。结果 术后2周：对照组与实验组无明显差异，无牙本质桥形成。术后4周：对照组无牙本质桥形成，实验组有部分骨样牙本质桥形成。术后8周：对照组牙髓组织基本正常，无牙本质桥形成，实验组有完整的骨样牙本质桥形成，其下方有排列整齐的成牙本质细胞样细胞形成。结论 首次发现hDSP在体内能够诱导牙髓细胞分化，形成修复性牙本质。     

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。     

人牙本质涎蛋白的抗体制备和组织表达研究

目的制备人牙本质涎蛋白抗体,观察其在不同组织的表达情况.方法用原核表达并经过初步纯化的人牙本质涎蛋白免疫新西兰大白兔获得多克隆抗血清并用Western blot法测定效价和检测牙胚和其它组织的蛋白表达.结果抗人牙本质涎蛋白抗血清效价可达1100000;Western blot显示牙本质涎蛋白在人牙胚中有表达,其分子量约为Mr 60×103左右.结论牙本质涎蛋白在人牙胚组织中有表达,提示其在牙齿发生、牙本质修复和再矿化中可能起重要作用.     

短期高浓度氟对小鼠磨牙胚中牙本质涎蛋白表达的影响

目的:研究短期高浓度氟对小鼠磨牙成牙本质细胞形态及牙本质涎蛋白(dentin sialoprotein, DSP)表达的影响,探讨氟对牙本质发育的作用机制.方法:选择4 d龄的ICR小鼠共32 只,随机分为2 组,每组16 只,各组中实验动物和对照动物各半.实验动物单次腹腔注射剂量分别为10 mg/kg体重和20 mg/kg体重的NaF,对照动物单次腹腔注射等剂量的NaCl,注射量均为10 μl/g, 24 h后处死动物.采用HE染色、免疫组化染色观察高浓度氟对小鼠磨牙不同分化阶段成牙本质细胞形态及DSP的表达,采用SPSS 13.0软件对数据进行分析.结果:实验组分泌期成牙本质细胞形态紊乱,正常的高柱状形态丧失,DSP的表达明显强于对照动物,差异有统计学意义(P<0.01),而成熟期成牙本质细胞未见明显变化.结论:短期高浓度氟能增强分泌期成牙本质细胞中DSP的表达,抑制成牙本质细胞的增殖分化及随后的基质合成与分泌,从而影响牙本质的发育.     

TGF-β1在氟中毒大鼠切牙牙髓中的表达

目的:研究过量氟对大鼠牙髓细胞TGF-β1表达的影响。方法:20只Wister大鼠,随机分为对照组和实验组。对照组用等量蒸馏水灌胃,实验组以20mg.kg-1.d-1氟化钠水灌胃。8周后处死动物,利用磨片、HE染色、免疫组化染色技术观察过量氟对大鼠切牙形态及TGF-β1表达的影响。采用SPSS10.0软件对数据进行t检验。结果:实验组大鼠切牙釉质牙本质生长线明显,球间牙本质增多,牙髓细胞及髓腔内侧牙本质TGF-β1表达显著弱于对照组,差异有显著性(P<0.01)。结论:过量氟可抑制牙髓细胞表达TGF-β1,影响牙体硬组织的结构。     

牙本质涎蛋白转基因小鼠的构建

目的 构建小鼠牙本质涎蛋白（DSP）转基因小鼠。方法 将pcDNA3．1载体中的CMV启动子替换为启动子cβ-actin，构建戴体pcDNA3．1-Cx。然后将PCR获得的DSP基因编码序列克隆到pcDNA3．1-Cx中。构建DSP转基因戴体peDNA3．1-Cx-dsp；将线性化的载体DNA注射到小鼠受精卵的雄原核，受精卵移植到假孕母鼠的输卵管。仔鼠出生后，用PCR及Southernblot检测阳性小鼠。结果 共移植717枚注射过的受精卵至29只受体鼠，移卵后产仔67只，阳性4只。阳性鼠分别传代。开始建系。结论 通过显微注射的方法成功获得了DSP转基因小鼠。     

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。     

牙本质涎蛋白转基因小鼠的建立和转基因表达的初步分析

目的:构建牙本质涎蛋白(DSP)转基因小鼠,并对转基因表达进行初步RT-PCR分析。方法:将pcDNA3.1载体中的CMV启动子替换为启动子cβ-actin,构建载体pcDNA3.1-CX,然后将PCR获得的DSP基因编码序列克隆到pcDNA3.1-CX中,构建DSP转基因载体pcDNA3.1-CX-dsp;将线性化的载体DNA注射到小鼠受精卵的雄原核,将受精卵移植到假孕母鼠的输卵管。仔鼠出生后,用PCR及Southern印迹检测阳性小鼠,并用RT-PCR对其中一小鼠的F1代进行转基因表达分析。结果:共移植717枚注射过的受精卵至29只受体鼠,移卵后产仔67只,阳性4只。检测到53号小鼠F1代外源性DSP的表达。结论:通过显微注射方法,成功获得了DSP转基因小鼠,并证实外源性DSP可在53号小鼠F1代获得表达。     

The dentin sialoprotein (DSP) expression in rat tooth germs following fluoride treatment: an immunohistochemical study

Fluoride is known to alter expression of dentin matrix proteins and affect their posttranslational modifications. OBJECTIVE: The objective of our study was to examine dentin sialoprotein (DSP) expression in the early and late bell stages of development of the first molar tooth germs in rats treated with fluoride. DESIGN AND METHODS: Pregnant dumps were divided into three groups. They were fed a standard diet and from the fifth day of pregnancy, each group received either tap water (with trace amounts of fluoride), tap water with a low concentration of fluoride, or tap water with a high concentration of fluoride. Changes in DSP expression and distribution were visualized by immunohistochemistry. RESULTS: Immunoreactivity for DSP was detected in the cervical regions of the early bell stage in tooth germs of the 1-day-old animals. The earliest reaction was visible in the control group and the group supplemented with the low fluoride concentration (F(L)) but not in the group supplemented with the high fluoride concentration (F(H)). In early bell stages across all experimental groups, the immunoreactivity to DSP was observed in the cusp tip regions and was localized to preameloblasts, young and mature odontoblasts, dental pulp cells, predentin, and dentin. Generally, more intense positive staining for DSP was detected in animals supplemented with the high fluoride concentration. In the late bell stage found in the 4-day-old control group and the group supplemented with the low fluoride concentration, immunoreactivity for DSP was less intense compared with younger animals. However, immunoreactivity was greater in the group treated with the high dose of fluoride. In this group, the positive immunostaining for DSP, especially in young ameloblasts, was prolonged and relatively strong. CONCLUSIONS: Fluoride supplementation causes changes in the developmental pattern of DSP expression and its distribution in rat tooth germs.     

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.     

过量氟对大鼠切牙釉丛蛋白表达的影响

目的研究过量氟对大鼠切牙发育过程中釉丛蛋白表达的影响,探讨氟斑牙的发病机制。方法选择20只Wistar大鼠,随机分成两组:对照组(蒸馏水组)和实验组(100mg/LF-)。复制大鼠氟斑牙模型。饲养8周处死动物,利用免疫组化和实时荧光定量PCR(Real-TimePCR)方法观察过量氟对大鼠切牙中釉丛蛋白表达的影响。结果免疫组化结果显示釉丛蛋白在分泌前期和分泌期的成釉细胞中呈阳性表达。实验组釉丛蛋白的表达明显弱于对照组,存在显著性差异(P<0.01)。Real-TimePCR结果显示釉丛蛋白mRNA在对照组表达明显高于在实验组表达水平(P<0.01)。结论过量氟可能通过抑制釉丛蛋白的表达,从而影响釉质的矿化,导致釉质发育障碍。     

Evidence for programmed odontoblast process retraction after dentine exposure in the rat incisor

ObjectiveTo re-examine the morphology and potential functions of odontoblasts in intact rat incisors and after cavity preparation into dentine.DesignIntact incisors were fixed, decalcified, snap frozen and sectioned (10 μm), before staining with rhodamine phalloidin or antibodies for cyto-skeletal proteins: vimentin and actin, ion transporter: NaK-ATPase, and dendritic cell marker: OX6. Samples with cavity were processed similarly and stained for actin and vimentin before comparing the lengths of odontoblast processes (OP) at baseline, 3 h and 24 h (n = 5 for each group).ResultsActin was expressed through the full length of OP, while vimentin immunoreactivity was not uniform, with 4 distinct regions. OP showed morphological complexity with fine branches emanating within different regions of dentine. Novel actin-positive tree-like OP were identified within predentine which reduced in intensity and length toward the incisal portion of the tooth. Specimens with cavities showed time-dependant pulpal retraction of OP.ConclusionsDifferences in structural antibody expression suggest functional variations in OP within different regions of dentine. The role of actin positive OP in predentine is not known, but could be related to dentine deposition, cellular stability or sensing mechanisms. Cavity preparation into dentine was followed by programmed retraction of OP which could be controlled either mechanically by the spatial limitation of the OP within dentinal tubules or structurally by the presence of vimentin, in addition to actin, in the mid-dentine.