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 如果要保护牙骨质、牙周膜，维持交界上皮 (JE) 至关重要 （PDL），并且牙槽骨是要实现的。随着我们发现 Wnt-，对 JE 屏障功能有了新的认识 乙脑中的反应性干细胞生态位。在本提案中，我们的目标是描述蜂窝参与者、利基信号和 控制和维持健康状态下以及损伤或疾病后乙脑干细胞生态位的调节机制。 AIM 1 实验将首先测试 Wnt/β-catenin 信号传导对于 JE 的维持是否是必需的。途径抑制将 使用表达可溶性 Wnt 抑制剂 Dkk1 的腺病毒在 Axin2LacZ/+ 小鼠中实现；对照将接受腺病毒 编码免疫球蛋白 G 的 Fc 部分。在规定的时间间隔内，定量分析将评估内源性 Wnt/β- 通过 Xgal 染色进行连环蛋白信号传导；乙脑半桥粒基因和蛋白质定量分布 免疫组织化学（qIHC）；通过 EdU/BrdU 标记观察 JE 和 GE 细胞周期动力学；以及炎症细胞浸润 通过 FACS 检测 JE 和 GE 下方的结缔组织。二、Wnt/β-catenin信号传导对于乙脑是否是必需的 再生将通过对 Wnt 谱系示踪剂（例如 Axin2CreERT2/+;R26RmTmG/+ 小鼠）进行部分牙龈切除术来确定 随后是 Ad-Dkk1/Ad-Fc 递送。谱系追踪和定量分析将建立 Wnt- 之间的关系 反应性细胞后代、细胞周期动力学、半桥粒基因和蛋白质分布以及乙脑屏障的再生 功能。 AIM 2 实验将评估 WNT 蛋白稳定制剂再生功能性乙脑的能力。合而为一 损伤修复模型 JE 将通过手术切除；在第二种模型中，JE 崩溃将通过结扎引起的触发 牙周炎;两者都将在 Axin2LacZ/+ 和 Axin2CreERT2/+;R26RmTmG/+ 小鼠中进行。 WNT 治疗遗嘱的交付 在多个时间点进行定量分析，以评估乙脑屏障功能的重建。 AIM 3 实验将表征 Wnt 反应性乙脑干细胞及其后代。 Wnt 反应性干细胞池 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 通过在规定的时间点收获 JE 和 GE 组织。 GFP+ 细胞将通过流式细胞术进行分选。基因表达谱 GFP+ 细胞将聚焦干细胞和分化标记物。荧光原位杂交将证实基因表达 使用与干细胞标记物、Wnt/β-catenin、Notch 和 Bone 成分相对应的 RNA 探针库进行模式 形态发生蛋白 (BMP) 途径。总的来说，该提案有望为以下问题提供重要的新见解： 维持乙脑干细胞生态位需要 Wnt/β-连环蛋白信号传导；调节 JE 和 GE 细胞增殖 差异化；并影响半桥粒介导的牙齿表面附着。另外，还应该解决 当前关于 JE 与 GE 干细胞库的分子特性及其分化潜力的争论。拟议的 这项工作还有可能确定一种创新的治疗策略来重建受损的乙型脑炎，从而开辟新的途径 牙周病后软组织附着恢复的途径。