Osteoimmunology of Retarded Bone Regeneration in Periodontitis

牙周炎骨再生迟缓的骨免疫学

• 批准号: 10451355
• 负责人: TOSHIHISA KAWAI
• 金额: $ 7.43万
• 依托单位: NOVA SOUTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451355
• 关键词: Administrative Supplement; Attenuated; Basic Science; Biological Assay; Bone Regeneration; Bone Resorption; Cell Culture Techniques; Cells; Coculture Techniques; Data; Data Analyses; Dental; Dentistry; Flow Cytometry; Future; Gifts; Goals; Grant; Hispanics; Knowledge; Mediating; Methods; Monitor; Monoclonal Antibodies; Mouth Diseases; Osteoclasts; Parents; Pathogenicity; Periodontitis; Porphyromonas gingivalis; Proteins; Reproducibility; Research Project Grants; Role; Science; Scientist; Statistical Data Interpretation; TNFSF11 gene; Training; Translational Research; Vesicle; Vimentin; career development; osteoclastogenesis; osteoimmunology; precursor cell; programs; receptor; responsible research conduct

This application for Diversity Administrative Supplement (PA-21-071) is written to request for the one-year support for Hispanic Post Bachelorette Trainee, Ms. Elizabeth Leon, under the parent RO1 grant, titled, “Osteoimmunology of regarded bone regeneration in periodontitis.” She will perform the basic research to elucidate the mechanism underlying the pathogenic bone resorption in periodontitis. Through the proposed training, she will not only become familiar with the operational approach to perform basic research related to the oral diseases, but also master the conceptual approach to find the knowledge gap and the way to solve the existing problem. The fundamentals for increasing the rigor and reproducibility, as well as the principle for responsible research conduct will be also learned. Parallelly, she will be working toward acceptance in the program of dentistry. The goal of this training is to support the career development of a future clinician/scientist in the basic and translational research in the dental sciences. Candidate’s research project background: Our unpublished data demonstrated that P. gingivalis’ vesicles can promote the osteoclastogenesis which was significantly attenuated by the vesicles isolated from PAD-KO P. gingivalis. Furthermore, P. gingivalis’ vesicles up-regulated the expression of OC-STAMP in the RANKL- stimulated RAW264.7 osteoclast precursor cells. Hypothesis: We hypothesized that PAD expressed by vesicles of P. gingivalis can citrullinate the host derived Vimentin which, in turn, acts on OC-STAMP expressed by osteoclasts and promotes osteoclastogenesis Project Objectives for Candidate: Objective 1: To evaluate the role of PAD expressed by vesicles of P. gingivalis (Pg vesicle-PAD) in citrullinating the host proteins, including Vimentin, in the co-culture of vesicles and osteoclasts. Objective 2: To examine whether Pg vesicle-PAD-mediated citrullination of host vimentin can alter Vimentin’s effect on promoting RANKL-induced osteoclastogenesis. The vesicles will be isolated from PAD-KO Pg W83 (gift from Dr. Potempa) and WT Pg W83 using a ultracentrifuge machine. Those isolated vesicles, as well as whole bacterial cells, will be co-cultured with osteoclast precursors. The level of citrullination of host proteins will be determined by W-blot and Mass-Spec. The expression of PAD by vesicles will be monitored using a W-blot. Finally, possible engagement of OC- STAMP expressed by osteoclasts which is the receptor for citrullinated Vimentin in osteoclastogenesis will be determined by blocking with anti-OC-STAMP mAb or anti-citrullinated Vimentin mAb. After completing these two objectives, the candidate will have mastered the methods of cell culture, W- blotting, qPCR, flow cytometry, and osteoclastogenesis assays, as well as basic statistical analysis and interpretation of data which strengthen her credential to be a candidate for graduate program.

本多样性行政补充申请 (PA-21-071) 是为了请求一年的 根据母公司 RO1 补助金，为西班牙裔后单身实习生 Elizabeth Leon 女士提供支持，标题为： “牙周炎中受关注的骨再生的骨免疫学。” 通过提出的方法阐明牙周炎致病性骨吸收的机制。 培训中，她不仅会熟悉进行相关基础研究的操作方法 口腔疾病，还要掌握概念方法，寻找知识差距和解决方法 提高严谨性和可重复性的基础，以及原则。 与此同时，她还将学习负责任的研究行为。 该培训的目标是支持未来临床医生/科学家的职业发展。 从事牙科科学的基础和转化研究。 候选人的研究项目背景：我们未发表的数据表明牙龈卟啉单胞菌的囊泡 可以促进破骨细胞生成，而从 PAD-KO 中分离的囊泡可显着减弱破骨细胞生成 此外，牙龈卟啉单胞菌的囊泡上调了 RANKL- 中 OC-STAMP 的表达。 刺激RAW264.7破骨细胞前体细胞。 假设：我们推测牙龈卟啉单胞菌囊泡表达的PAD可以使宿主衍生的瓜氨酸化 波形蛋白反过来作用于破骨细胞表达的 OC-STAMP 并促进破骨细胞生成 候选人的项目目标： 目的 1：评估牙龈卟啉单胞菌囊泡（Pg vesicle-PAD）表达的 PAD 在 在囊泡和破骨细胞的共培养中对宿主蛋白（包括波形蛋白）进行瓜氨酸化。 目标 2：检查 Pg 囊泡-PAD 介导的宿主波形蛋白瓜氨酸化是否可以改变 Vimentin 对促进 RANKL 诱导的破骨细胞生成的作用。 将使用 PAD-KO Pg W83（Potempa 博士赠送）和 WT Pg W83 分离囊泡。 这些分离的囊泡以及整个细菌细胞将与超速离心机共培养。 破骨细胞前体的瓜氨酸化水平将通过W-印迹和质谱测定。 将使用W-印迹监测囊泡的PAD表达最后，OC-的可能参与。 破骨细胞表达的 STAMP 是破骨细胞生成中瓜氨酸波形蛋白的受体 通过使用抗 OC-STAMP mAb 或抗瓜氨酸 Vimentin mAb 封闭来确定。 完成这两个目标后，候选人将掌握细胞培养的方法，W- 印迹、qPCR、流式细胞术和破骨细胞生成测定，以及基本统计分析和 对数据的解释增强了她作为研究生项目候选人的资格。