Analysis of GFP Transgenes in Odontoblast in Differentiation

描述（由申请人提供）：牙本质发生受源自牙乳头的单层高度分化的有丝分裂后成牙本质细胞调节。尽管在发现调控牙齿发生和形成的关键分子和信号通路方面取得了重大进展，但导致牙本质形成的机制相对未知。这部分是由于难以获得用于分子分析的祖细胞的同质群体以及缺乏用于鉴定成牙本质细胞分化的中间阶段的合适分子标记。本提案的总体目标是使用启动子-GFP报告基因转基因小鼠作为一种新的实验模型，以解开调节牙髓祖细胞线性逐步进展为成牙本质细胞的潜在分子机制。该提案将使用Col 1a 1-GFP转基因的激活，通过FACS分选在分化的早期阶段鉴定和分离细胞，用于进一步的细胞和分子分析，包括检查其分化潜力和基因表达谱。提出了三个具体目标。目的#1将检验Col 1a 1-GFP转基因的阶段特异性激活可以作为标记物来区分和鉴定牙髓细胞亚群以用于体外进一步谱系分析的假设。目的#2将检验胰岛素样生长因子（IGF-I）刺激成牙本质细胞分化的假设。在目标2中，Col 1a 1-GFP转基因将被用于检查IGF-I刺激成牙本质细胞分化的潜在机制。在这个目标中，IGF-I水平的改变对牙髓细胞增殖、凋亡和分化的影响来自于骨/成牙本质细胞靶向IGF-I过表达的转基因小鼠（pOBCo！3.6-IGF）和IGF-I单倍不足（Igf 1，HET）的转基因小鼠。目的#3将检验Col 1a 1-GFP转基因可用作标记物的假设，以在异位部位的体内移植测定中评估粘附牙髓成纤维细胞的牙本质形成潜力。可以预见，从这些实验中获得的结果将为开发基于干细胞的治疗方法提供基本见解，用于修复和再生牙本质，并最终修复和再生因先天性疾病，疾病和损伤而受损的整个牙齿。

DESCRIPTION (provided by applicant): Dentinogenesis is regulated by a single layer of highly differentiated post-mitotic odontoblasts originating from the dental papilla. Despite significant progress in the discovery of key molecules and signaling pathways regulating tooth initiation and patterning, the mechanisms leading to dentinogenesis are relatively unknown. This has been due, in part, to difficulties in obtaining homogenous populations of progenitor cells for molecular analysis and the lack of suitable molecular markers for identifying the intermediate stages of odontoblast differentiation. The overall goals of this proposal are to use promoter-GFP reporter transgenic mice as a novel experimental model to unravel the underlying molecular mechanisms regulating the linear stepwise progression of pulp progenitor cells into odontoblasts. This proposal will use the activation of Col1a1-GFP transgenes to identify and isolate cells at early stages of differentiation by FACS sorting for further cellular and molecular analyses including an examination of their differentiation potential and gene expression profile. 3 specific aims are proposed. Aim #1 will test the hypothesis that stage-specific activation of Col1a1-GFP transgenes can serve as markers to distinguish and identify sub-populations of cells from dental pulp for further lineage analysis in vitro. Aim #2 will test the hypothesis that insulin-like growth factor (IGF-I) stimulates odontoblast differentiation. In Aim 2, Col1a1-GFP transgenes will be used to examine the underlying mechanisms by which IGF-I stimulates odontoblast differentiation. In this aim the effects of the altered levels of IGF-I on proliferation, apoptosis, and differentiation of pulp cells derived from transgenic mice with bone/odontoblast targeted over-expression of IGF-I (pOBCo!3.6-IGF), and transgenic mice with IGF-I haploinsufficiency (Igf1, HET) will also be examined. Aim #3 will test the hypothesis that Col1a1-GFP transgenes can be used as a marker to assess the dentin forming potential of adherent pulp fibroblasts in an in vivo transplantation assay at an ectopic site. It can be envisioned that results obtained from these experiments, will provide fundamental insights in the development of stem cell-based treatments for the repair and regeneration of dentin and eventually whole tooth that has been compromised by congenital disorders, diseases, and injuries.