GENETIC EXPRESSION OF HUMAN ODONTOBLASTS

成牙本质细胞是一种独特的细胞类型，负责牙本质有机基质的加工。最初在牙齿形态发生中起关键作用，它在保护和维持完全形成的牙齿中同样不可或缺。传统上，由于其高度分化和有丝分裂后状态，这种细胞不适合简单的实验模型。成牙本质细胞的部分特征已完成使用生化，显微镜，最近，分子技术，主要是在啮齿动物系统。然而，缺乏对人类成牙本质细胞的完整表征。这个建议的总体目标是建立一个模型，通过这个模型可以鉴定出静止期和刺激期成牙本质细胞的成牙本质细胞特异性转录本。 我们建议通过人成牙本质细胞mRNA和原代人成骨细胞mRNA之间的消减杂交来实现这一点。将通过斑点杂交和北方印迹分析鉴定独特的转录物，并通过序列分析表征。 独特的转录本可以作为探针原位杂交，以验证其在各种组织中的表达范围。已证实的成牙本质细胞特异性转录本将被用作探针，筛选人类基因组文库中所选克隆的完整基因序列，以研究基因加工和调控，从而描述遗传调控在修复性牙本质形成中的作用。 未来的研究将包括利用成牙本质细胞特异性转录本作为分化标志物来鉴定祖细胞库以及应用所提出的技术来研究人类成牙本质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