DENTIN SPECIFIC PHOSPHOPROTEINS AND MINERALIZATION

本项目的目标是研究这些细胞的生物学功能。 牙本质形成过程中的牙本质特异性磷蛋白 生物矿化，从而促进了我们对 成牙本质细胞分化和牙本质形成的过程。这个目标是 基于一组新出现的文献显示酸性蛋白质 通常在晶体形成的直接调节中起关键作用。我们的 假说是牙本质特异的磷蛋白DMP-1和DPP(S)， 是成牙本质细胞分化的标志，在 牙本质中羟基磷灰石形成的成核与调控 细胞外基质矿化。我们的首要目标是隔离 并鉴定了DMP-1和DPP的全长cDNA(S)。这将是 通过构建和筛选小鼠磨牙cDNA来实现 图书馆。一旦确定，DMP-1和DPP(S)的cDNA将被 在原核和真核宿主中异源表达，以及 用于产生特定抗体的纯化蛋白。这些抗体 将用于映射DMP-1和DMP-1的顺序表达模式 牙本质细胞外与成牙本质细胞分化相关的DPP(S) 基质矿化采用免疫组织化学方法。的表达方式 这些牙本质特异的磷蛋白在转录水平上将 也可以通过RT-PCR扩增和原位杂交进行检测 技巧。最后，DMP-1和DPP(S)的生物学功能将 利用反义“基因敲除”和抗体扰动进行研究 监测产生的矿物相和基质形成的策略 利用电子显微镜、X射线衍射仪和电子探针 分析。这些实验将利用一种独特的单层 本实验室建立成牙本质细胞培养体系 确定牙本质特异性磷酸蛋白的功能。这 文化体系此前已被证明有利于 牙乳头间充质细胞向成牙本质细胞分化， 维持成牙本质细胞的表型、顺序表达和 牙本质细胞外基质蛋白的分泌和牙本质 生物矿化作用。此外，我们最近还建立了 永生化小鼠成牙本质细胞和牙乳头间充质细胞系 在成牙本质细胞分化的关键阶段，这将用于 确定牙本质特异性磷蛋白的调节作用 在DECM产生和随后的矿化上的表达。这两个 将使用关键的研究工具来研究生物功能 成牙本质细胞分化及成牙本质细胞分化过程中牙本质特异性磷蛋白的表达 牙本质矿化在这方面的应用。

The goal of this project is to study the biological function of these dentin-specific phosphoproteins in the process of dentin biomineralization, thereby advancing our overall understanding of the process of odontoblast differentiation and dentinogenesis. This goal is based on an emerging body of literature showing that acidic proteins generally play a key role in direct regulation of crystal formation. Our hypothesis is that the dentin-specific phosphoproteins, DMP 1 and DPP(s), are markers for odontoblast differentiation which are important in the nucleation and regulation of hydroxyapatite formation during dentin extracellular matrix mineralization. Our first objective is to isolate and characterize the full-length cDNAs for DMP-1 and DPP(s). This will be accomplished by construction and screening of a mouse molar cDNA library. Once identified, the DMP-1 and DPP(s) cDNAs will be heterologously expressed in procaryotic and eukaryotic hosts, and the purified protein used to produce specific antibodies. These antibodies will be used to map the sequential expression patterns of DMP-1 and DPP(s) during odontoblast differentiation related to dentin extracellular matrix mineralization using immunohistochemistry. The expression of these dentin-specific phosphoproteins at a transcriptional level will also be examined using RT-PCR amplification and in situ hybridization techniques. Finally, the biological function of DMP-1 and DPP(s) will be investigated using antisense "knockout" and antibody perturbation strategies monitoring the resulting mineral phase and matrix formation using electron microscopy, X-ray diffraction and electron microprobe analyses. These experiments will take advantage of a unique monolayer odontoblast cell culture system established in our laboratory to determine the functions of the dentin-specific phosphoproteins. This culture system has previously been shown to be conducive for the cytodifferentiation of odontoblasts from dental papilla mesenchyme cells, maintenance of the odontoblast phenotype, sequential expression and secretion of dentin extracellular matrix proteins, and dentin biomineralization. In addition, we have recently established immortalized mouse odontoblast and dental papilla mesenchyme cell lines at key stages of the odontoblast differentiation which will be used to determine the effect of modulation of dentin-specific phosphoprotein expression on DECM production and subsequent mineralization. These two key research tools will be used to investigate the biological function of dentin-specific phosphoproteins during odontoblast differentiation and dentin mineralization in this application.