Molecular mechanisms mediating the soft tissue attachment to teeth

介导软组织附着到牙齿的分子机制

• 批准号: 10588063
• 负责人: Jill Helms A Helms
• 金额: $ 70.61万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-20 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10588063
• 关键词: Address; Adenoviruses; Anatomy; Biochemical; Bone Morphogenetic Proteins; Bromodeoxyuridine; Cell Cycle; Cell Cycle Kinetics; Cell Differentiation process; Cells; Clinical; Connective Tissue; Data; Dental Cementum; Differentiation Antigens; Disease; Embryo; Epithelial Attachment; Epithelial Cell Junction; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; Excision; Exposure to; Flow Cytometry; Fluorescent in Situ Hybridization; Formulation; Gene Expression Profile; Gene Expression Profiling; Genes; Gingiva; Gingivectomy; Goals; Harvest; Health; Hemidesmosomes; Heterogeneity; Histology; Homeostasis; Human; Immunoglobulin G; Immunohistochemistry; Infiltration; Inflammatory; Injury; Jaw; Knowledge; Label; Libraries; Ligature; Maintenance; Mediating; Modeling; Molecular; Mus; Natural regeneration; Operative Surgical Procedures; Oral; Oral cavity; Pathway interactions; Periodontal Diseases; Periodontal Ligament; Periodontitis; Placebos; Population; Prevention; Prevention strategy; Proliferating; RNA Probes; Recombinants; Regenerative capacity; Reporter; Research Personnel; Role; Series; Signal Transduction; Site; Skeleton; Sorting; Source; Stains; Tamoxifen; Testing; Therapeutic; Tissues; Tooth structure; Tracer; WNT Signaling Pathway; Wnt proteins; Wnt-3A protein; Work; alveolar bone; beta catenin; epithelial stem cell; epithelium regeneration; experimental study; inhibitor; injury and repair; innovation; insight; lipid nanoparticle; new therapeutic target; notch protein; novel therapeutic intervention; preservation; protein distribution; repair model; restoration; self-renewal; soft tissue; stem cell biomarkers; stem cell niche; stem cell self renewal; stem cells; targeted treatment; therapeutic protein; tooth surface; wound healing

Project abstract Maintaining the junctional epithelium (JE) is of primary importance if preservation of the cementum, periodontal ligament (PDL), and alveolar bone is to be achieved. New insights into JE barrier functions came with our discovery of a Wnt- responsive stem cell niche in the JE. In this proposal, our goal is to characterize the cellular players, niche signals, and regulatory mechanisms that control and maintain the JE stem cell niche in health, and after damage or disease. AIM 1 experiments will first test whether Wnt/β-catenin signaling is necessary for JE maintenance. Pathway inhibition will be achieved in Axin2LacZ/+ mice using adenovirus expressing the soluble Wnt inhibitor Dkk1; controls will receive adenovirus encoding the Fc portion of immunoglobulin G. At defined intervals, quantitative analyses will assess endogenous Wnt/β- catenin signaling via Xgal staining; JE hemidesmosomal gene and protein distribution via quantitative immunohistochemistry (qIHC); JE and GE cell cycle kinetics by EdU/BrdU labeling; and inflammatory cell infiltration in connective tissues underlying the JE and GE by FACS. Second, whether Wnt/β-catenin signaling is necessary for JE regeneration will be determined by subjecting Wnt lineage tracer e.g., Axin2CreERT2/+;R26RmTmG/+ mice to partial gingivectomy followed by Ad-Dkk1/Ad-Fc delivery. Lineage-tracing and quantitative analyses will establish a relationship between Wnt- responsive cell progeny, cell cycle kinetics, hemidesmosomal gene and protein distribution, and regeneration of JE barrier functions. AIM 2 experiments will evaluate the ability of a stabilized formulation of WNT protein to regenerate a functional JE. In one injury-repair model the JE will be surgically excised; in a second model, JE breakdown will be triggered via a ligature-induced periodontitis; both will be carried out in Axin2LacZ/+ and Axin2CreERT2/+;R26RmTmG/+ mice. Delivery of the WNT therapeutic will be followed at multiple timepoints by quantitative analyses to assess re-establishment of JE barrier functions. AIM 3 experiments will characterize Wnt-responsive JE stem cells and their progeny. Wnt-responsive stem cell pools from adjacent gingival epithelium (GE) will serve as control. Axin2CreERT2/+;R26RmTmG/+ mice will be exposed to tamoxifen, followed by harvest of JE and GE tissues at defined timepoints. GFP+ cells will be sorted by flow cytometry. Gene expression profiling of GFP+ cells will focus stem cell and differentiation markers. Fluorescent in situ hybridization will confirm gene expression patterns using RNA probe libraries corresponding to stem cell markers, components of Wnt/β-catenin, Notch, and Bone Morphogenetic Protein (BMP) pathways. Collectively, this proposal promises to provide important new insights into the requirement for Wnt/β-catenin signaling in maintaining the JE stem cell niche; regulating JE and GE cell proliferation and differentiation; and influencing hemidesmosomal-mediated attachment to the tooth surface. In addition, it should resolve the current debate over the molecular identities of JE v. GE stem cell pools and their differentiation potential. The proposed work also has the potential to identify an innovative therapeutic strategy for rebuilding a damaged JE and thus open new avenues for the restoration of the soft tissue attachment following periodontal diseases.

项目摘要 如果保存牙周韧带，保持连接上皮（JE）至关重要 （PDL），并且将实现大骨骨。我们发现Wnt的新见解。 JE中的反应性干细胞生态位。在此提案中，我们的目标是表征蜂窝播放器，利基信号和 控制和维持健康中的JE干细胞生态位，以及损害或疾病后的调节机制。 AIM 1实验将首先测试Wnt/β-catenin信号是否需要用于维持JE。途径抑制作用 使用表达固体Wnt抑制剂DKK1的腺病毒在AXIN2LACZ/+小鼠中实现；控件将收到腺病毒 编码免疫球蛋白G的FC部分。在定义的间隔中，定量分析将评估内源性Wnt/β- Catenin信号通过Xgal染色； JE Hemidesmosamal基因和蛋白质分布 免疫组织化学（QIHC）; JE和GE细胞周期动力学通过EDU/BRDU标记；和炎症细胞浸润 FACS将JE和GE的结缔组织。其次，JE是否需要Wnt/β-catenin信号传导 再生将通过对Wnt谱系示踪剂（例如AXIN2CREERT2/+; R26RMTMG/+小鼠）进行确定 然后进行AD-DKK1/AD-FC交付。谱系追踪和定量分析将建立Wnt-之间的关系 反应敏感细胞后代，细胞周期动力学，半底膜基因和蛋白质分布以及JE屏障的再生 功能。 AIM 2实验将评估Wnt蛋白稳定公式再生功能性JE的能力。一个 损伤修复模型将通过手术超出JE；在第二个模型中，将通过结扎的诱导触发JE分解 牙周炎；两者都将在AXIN2LACZ/+和AXIN2CREERT2/+; R26RMTMG/+小鼠中进行。 WNT治疗的交付将 在多个时间点上进行定量分析，以评估重新建立JE屏障功能。 AIM 3实验将表征Wnt响应的JE干细胞及其后代。 Wnt响应的干细胞池 邻近的牙龈上皮（GE）将作为对照。 AXIN2CREERT2/+; R26RMTMG/+小鼠将暴露于他莫昔芬，然后接触 通过在定义的时间点上收获JE和GE组织。 GFP+细胞将通过流式细胞仪进行排序。基因表达分析 GFP+细胞的组合将聚焦干细胞和分化标记。荧光原位杂交将确认基因表达 使用对应于干细胞标记的RNA探针库，Wnt/β-catenin，Notch和Bone的组成部分的模式 形态发生蛋白（BMP）途径。总的来说，该提案有望为 Wnt/β-catenin信号传导的需求在维持JE干细胞生态位；调节JE和GE细胞增殖以及 分化；并影响半体体介导的牙齿表面附着。另外，它应该解决 当前关于JEv。GE干细胞池的分子身份及其分化潜力的争论。提议 工作还具有确定创新的理论策略来重建损坏的JE并因此开放新的策略 牙周疾病后恢复软组织附着的途径。