GENETIC EXPRESSION OF HUMAN ODONTOBLASTS

成牙细胞是一种独特的细胞类型，它负责形成牙本质的有机基质。它最初在牙齿的形态发生中起着关键作用，在保护和维持完全形成的牙齿方面也同样不可或缺。由于其高度分化和有丝分裂后的状态，传统上这种细胞不适合简单的实验模型。主要在啮齿动物系统中，利用生物化学、显微镜和最近的分子技术已经完成了成牙细胞的部分表征。然而，对人类成牙细胞的完整描述是缺乏的。本建议的总体目标是建立一种模型，通过这种模型可以识别静止和受刺激的成牙细胞的成牙细胞特异性转录本。我们建议通过在人成牙细胞mRNA和原代人成骨细胞mRNA之间进行减法杂交来实现这一点。独特的转录本将通过点杂交和Northern杂交分析鉴定，并通过序列分析鉴定。然后，独特的转录本可以用作原位杂交探针，以验证其在各种组织中的表达范围。确定的成牙本质特异性转录本将作为探针，筛选人类基因组文库，以获得所选克隆的完整基因序列，研究基因加工和调控，从而描述基因调控在修复性牙本质发生中的作用。未来的研究将包括利用成牙细胞特异性转录本作为鉴定祖细胞池的分化标记，以及应用所提出的技术研究人类成牙细胞的不同激活状态

The odontoblast is a unique cell type which is responsible for the elaboration of the organic matrix of dentin. Initially pivotal in tooth morphogenesis, it becomes equally as integral in the protection and maintenance of the fully formed tooth, Traditionally this cell has not been amenable to simplistic experimental models due to its highly differentiated and post-mitotic state. Partial characterization of the odontoblast has been accomplished using biochemical, microscopic and, most recently, molecular techniques, predominantly in rodent systems. A full characterization of the human odontoblast, however, is lacking., The overall aim of this proposal is to develop a model whereby odontoblast- specific transcripts may be identified for both quiescent and stimulated odontoblasts. We propose to do this by performing subtractive hybridization between human odontoblast mRNA and that of primary human osteoblasts. Unique transcripts will be identified by dot blot hybridization and Northern blot analysis and characterized by sequence analysis. Unique transcripts can then be used as probes in situ hybridization to verify their scope of expression in a variety of tissues. Confirmed odontoblast specific transcripts will be used as probes to screen human genomic libraries for the complete gene sequence of selected clones to study gene processing and regulation and thereby describe the role of genetic regulation in reparative dentinogenesis. Future studies would include the utilization of odontoblast-specific transcripts as differentiation markers for identifying progenitor cell pools as well as application of the proposed technique to study different activation states of human odontoblasts