Molecular mechanisms mediating the soft tissue attachment to teeth

父R01摘要 保留牙骨质、牙周膜和牙槽骨的关键是保持牙髓上皮(JE)。 对乙脑屏障功能的新见解来自于我们在乙脑中发现了WntResponse干细胞利基。在这份提案中， 我们的目标是描述控制和维护乙脑的手机玩家、利基信号和调节机制的特征。 干细胞在健康中的位置，以及年老的损伤或疾病。目标1实验将测试WNT/β-连环蛋白信号是否 乙脑维护所必需的。通过腺病毒表达可溶性腺病毒可在Axin2LacZ/+小鼠体内实现途径抑制 WNT抑制剂Dkk1；对照组将接受编码免疫球蛋白G的Fc启动子的腺病毒分析 将通过XGAL染色评估内源性Wnt/β连环蛋白信号；JE半桥粒基因和蛋白通过 免疫组织化学(QIHC)；EDU/BrdU标记JE和GE细胞周期蛋白；fl炎症细胞 在fi中，JE和GE的连接中的LTRA-ON由流式细胞仪检测。第二，WNT/β-连环蛋白信号通路是否对 JE再生将通过以下方式确定：将Wnt谱系示踪剂，例如Axin2CreERT2/+；R26RmTmG/+小鼠替换为Par-al 牙龈切除后植入Ad-Dkk1/Ad-Fc。血统追踪和量化分析将建立一种关系 Wnt反应细胞后代、细胞周期因子、半染色体基因和蛋白质分布与再生 我的屏障功能。目的2实验将评估WNT蛋白稳定配方的再生能力 一种功能性的JE。在一种损伤修复模型中，乙脑将被手术切除；在第二种模型中，乙脑的破裂将被触发。 通过结扎诱导的膜-S；这两种方法将在Axin2LacZ/+和Axin2CreERT2/+；R26RmTmG/+小鼠身上进行。递送 将遵循WNT疗法-C来评估乙脑屏障功能的重建。目标3实验将描述 对WNT反应的乙脑干细胞及其后代。来自邻近牙龈上皮(GE)的WNT反应干细胞库将 充当控制者。Axin2CreERT2/+；R26RmTmG/+小鼠将暴露于他莫昔芬，随后在 DefiNed-meints.绿色荧光蛋白阳性细胞将通过fl流式细胞仪进行分选。绿色荧光蛋白+细胞的基因表达促进fi将聚焦干细胞 和DiffEren-a-on记号笔。荧光原位杂交法利用fi探针库控制RM型基因表达 与干细胞标记物相对应的Wnt/β-连环蛋白、Notch和骨形态发生蛋白的组成 小路。综上所述，这项提案有可能识别一种重建受损房屋的创新疗法-c战略 JE，为牙周病后牙周粘连的修复开辟了新的途径。 拟议补编 乙脑是抵抗细菌感染的重要屏障，而细菌感染在预防牙周病中是至关重要的。在这 建议的多样性补充，我们将希望阐明WNT/β-连环蛋白信号在维持屏障和 乙脑的附属功能。扩展的AIM 2实验将使用Wnt缺陷的转基因小鼠模型 信号，例如Ocn-Cre；Wlsfl/fl，以评估乙脑和Wnt反应干细胞生态位是否被破坏。牙槽骨 fl炎性细胞反应和JE半桥粒蛋白通过qIHC； TRAP/ALP检测骨改建活性。这项提议有可能推进WNT疗法-c治疗 对于牙周病，然后可以使贫困社区更统一地获得这些疾病。

Summary of Parent R01 Maintaining the junc-onal epithelium (JE) is essen-al to preserve cementum, periodontal ligament (PDL), and alveolar bone. New insights into JE barrier func-ons came with our discovery of a Wntresponsive 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 aGer damage or disease. AIM 1 experiments will test whether Wnt/β-catenin signaling is necessary for JE maintenance. Pathway inhibi-on will be achieved in Axin2LacZ/+ mice using adenovirus expressing the soluble Wnt inhibitor Dkk1; controls will receive adenovirus encoding the Fc por-on of immunoglobulin G. Quan-ta-ve analyses will assess endogenous Wnt/βcatenin signaling via Xgal staining; JE hemidesmosomal gene and protein distribu-on via quan-ta-ve immunohistochemistry (qIHC); JE and GE cell cycle kine-cs by EdU/BrdU labeling; and inflammatory cell infiltra-on in connec-ve -ssues underlying the JE and GE by FACS. Second, whether Wnt/β-catenin signaling is necessary for JE regenera-on will be determined by subjec-ng Wnt lineage tracer e.g., Axin2CreERT2/+;R26RmTmG/+ mice to par-al gingivectomy followed by Ad-Dkk1/Ad-Fc delivery. Lineage-tracing and quan-ta-ve analyses will establish a rela-onship between Wntresponsive cell progeny, cell cycle kine-cs, hemidesmosomal gene and protein distribu-on, and regenera-on of JE barrier func-ons. AIM 2 experiments will evaluate the ability of a stabilized formula-on of WNT protein to regenerate a func-onal 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 periodon--s; both will be carried out in Axin2LacZ/+ and Axin2CreERT2/+;R26RmTmG/+ mice. Delivery of the WNT therapeu-c will be followed to assess re-establishment of JE barrier func-ons. 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 -ssues at defined -mepoints. GFP+ cells will be sorted by flow cytometry. Gene expression profiling of GFP+ cells will focus stem cell and differen-a-on markers. Fluorescent in situ hybridiza-on will confirm gene expression pacerns using RNA probe libraries corresponding to stem cell markers, components of Wnt/β-catenin, Notch, and Bone Morphogene-c Protein (BMP) pathways. Collec-vely, this proposal has the poten-al to iden-fy an innova-ve therapeu-c strategy for rebuilding a damaged JE and thus open new avenues for the restora-on of the soG -ssue acachment following periodontal diseases. Proposed Supplement The JE acts as an essen-al barrier against bacterial infec-on, which is crucial in preven-ng periodontal diseases. In this proposed diversity supplement, we will look to elucidate the role of Wnt/β-catenin signaling in maintaining the barrier and acachment func-ons of the JE. Extended Aim 2 experiments will use a transgenic mouse model with a defect in Wnt signaling, e.g., OCN-Cre;Wlsfl/fl, to evaluate if the JE and Wntresponsive stem cell niche become disrupted. Alveolar bone morphology will be assessed by µCT tomography; inflammatory cell response and JE hemidesmosomal proteins via qIHC; and bone remodeling ac-vity by TRAP/ALP assays. This proposal has the poten-al to advance Wnt therapeu-c treatments for periodontal diseases, which can then be made more uniformly accessible to disadvantaged communi-es.