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 保持连接上皮（JE）是首要的，如果保存牙骨质，牙周膜， (PDL)和牙槽骨。我们发现了一种Wnt- 在乙脑中的反应性干细胞龛。在这个建议中，我们的目标是描述细胞的球员，利基信号， 调控机制，控制和维持乙脑干细胞生态位的健康，损伤或疾病后。 AIM 1实验将首先测试Wnt/β-连环蛋白信号传导是否是维持JE所必需的。通路抑制将 使用表达可溶性Wnt抑制剂Dkk 1的腺病毒在Axin 2LacZ/+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，定量分析将评估内源性Wnt/β- 通过Xgal染色的连环蛋白信号传导;通过定量分析的JE半桥粒基因和蛋白质分布 免疫组织化学（qIHC）;通过EdU/BrdU标记的JE和GE细胞周期动力学;以及在结肠癌中的炎性细胞浸润。 JE和GE下面的结缔组织通过FACS。第二，Wnt/β-catenin信号通路是否是乙脑所必需的 再生将通过使Wnt谱系示踪剂，Axin 2CreERT 2/+; R26 RmTmG/+小鼠进行部分牙龈切除术 随后进行Ad-Dkk 1/Ad-Fc递送。谱系追踪和定量分析将建立Wnt- 应答细胞后代、细胞周期动力学、半桥粒基因和蛋白分布以及乙脑屏障的再生 功能协调发展的 AIM 2实验将评估WNT蛋白的稳定制剂再生功能性JE的能力。在一个 在损伤修复模型中，将通过手术切除JE;在第二个模型中，将通过结扎诱导的 牙周炎;两者都将在Axin 2LacZ/+和Axin 2CreERT 2/+; R26 RmTmG/+小鼠中进行。WNT治疗意愿的交付 在多个时间点进行定量分析，以评估JE屏障功能的重建。 AIM 3实验将表征Wnt响应性JE干细胞及其后代。Wnt反应性干细胞池来自 相邻的牙龈上皮（GE）将作为对照。Axin 2CreERT 2/+; R26 RmTmG/+小鼠将暴露于他莫昔芬，随后 在规定的时间点收获JE和GE组织。将通过流式细胞术分选GFP+细胞。基因表达谱 GFP+细胞将聚焦于干细胞和分化标志物。荧光原位杂交将证实基因表达 使用对应于干细胞标志物、Wnt/β-连环蛋白、Notch和Bone的组分的RNA探针文库的模式 形态发生蛋白（BMP）通路。总的来说，这一建议有望为解决全球气候变化问题提供重要的新见解。 Wnt/β-连环蛋白信号传导在维持JE干细胞生态位中的需要;调节JE和GE细胞增殖， 分化;和影响半桥粒介导的附着到牙齿表面。此外，还应解决 目前关于乙脑与通用电气干细胞库的分子身份及其分化潜力的争论。拟议 这项工作也有可能确定一种创新的治疗策略，用于重建受损的乙脑，从而开辟新的 牙周病后软组织附着的恢复途径。