Analysis of GFP Transgenes in Odontoblast in Differentiation

描述（由申请人提供）：牙本质形成是由源自牙乳头的单层高度分化的有丝分裂后成牙细胞调控的。尽管在发现调节牙齿形成和模式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。这在一定程度上是由于难以获得同源祖细胞群体进行分子分析，以及缺乏合适的分子标记来识别成牙髓细胞分化的中间阶段。本研究的总体目标是利用启动子- gfp报告基因转基因小鼠作为一种新的实验模型，揭示髓质祖细胞线性逐步向成牙髓细胞发展的潜在分子机制。本提案将利用Col1a1-GFP转基因的激活，通过FACS分选鉴定和分离处于分化早期阶段的细胞，以进一步进行细胞和分子分析，包括检查其分化潜力和基因表达谱。提出了3个具体目标。目的1将验证Col1a1-GFP转基因的阶段特异性激活可以作为区分和识别牙髓细胞亚群的标记，以便在体外进一步进行谱系分析的假设。目的2将验证胰岛素样生长因子（IGF-I）刺激成牙细胞分化的假设。在aims 2中，Col1a1-GFP转基因将用于检查IGF-I刺激成牙细胞分化的潜在机制。在这个目的中，IGF-I水平的改变对牙髓细胞增殖、凋亡和分化的影响也将被研究，这些牙髓细胞来源于骨/牙髓细胞靶向过表达IGF-I的转基因小鼠（pOBCo!3.6-IGF）和IGF-I单倍不足的转基因小鼠（Igf1， HET）。Aim #3将验证Col1a1-GFP转基因可以作为一种标记物，在异位部位的体内移植试验中评估贴壁牙髓成纤维细胞形成牙本质的潜力。可以预见，从这些实验中获得的结果将为基于干细胞的治疗的发展提供基本的见解，用于修复和再生牙本质，并最终修复因先天性疾病、疾病和损伤而受损的整颗牙齿。

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.