Evaluation of Osteocyte Dedifferentiation Process

骨细胞去分化过程的评价

• 批准号: 7874902
• 负责人: IVO Kalajzic
• 金额: $ 17.21万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7874902
• 关键词: Ablation; Adult; Affect; Automobile Driving; Bone Matrix; Bone Resorption; Bone remodeling; Cell Line; Cell Lineage; Cells; Characteristics; Data; Disease; Evaluation; Excision; Exhibits; Exposure to; Flow Cytometry; Foundations; Functional disorder; Future; Gene Expression; Genetic Recombination; Goals; Grant; Hormones; In Vitro; Individual; Inhibition of Apoptosis; Knowledge; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Osteoblasts; Osteocytes; Osteogenesis; Osteoporosis; PTH gene; Parathyroid Hormones; Pharmaceutical Preparations; Population; Process; Proliferating; Property; Protocols documentation; RNA; Regulation; Reporter; Side; Source; Staging; Stem cells; Testing; Therapeutic Agents; Tissues; Transgenes; Transgenic Mice; Transgenic Model; Transgenic Organisms; Transplantation; Visual; bone; bone mass; dentin matrix protein 1; design; human PTH protein; in vivo; in vivo Model; novel; osteoblast differentiation; osteoprogenitor cell; programs; promoter; public health relevance; recombinase; repaired; response; self-renewal

DESCRIPTION (provided by applicant): Presently, there is no evidence for the ability of mature osteoblast lineage cells to dedifferentiate. However, if possible, de-differentiation of mature osteoblast lineage cells could provide an additional source of osteoblast population capable of proliferation and differentiation. We hypothesize that under the conditions of high demand for osteoblast lineage activity, preosteocytes and osteocytes can undergo a de-differentiation, accompanied by dramatic changes in gene expression. This process would generate cells that could exhibit proliferation potential, followed by differentiation into bone matrix producing osteoblasts. Our preliminary data support these hypotheses, and in this proposal we seek to determine whether de-differentiation of osteoblast lineage cells is an integral aspect of bone remodeling and to evaluate the changes in gene expression involved. Intermittent administration of PTH results in a bone-anabolic response, but the mechanisms involved are not completely understood. We hypothesize that PTH-induced de-differentiation of osteoblast lineage cells could contribute to this bone-anabolic response. To test these hypotheses, we propose an in vivo evaluation of dedifferentiation - redifferentiation of preosteocytes/osteocytes. Utilizing a transgenic model in which we can achieve a complete ablation of mature osteoblasts, we will evaluate the ability of de-differentiated preosteocytes/osteocytes to repopulate the osteoblast niche. In addition, the ability of de-differentiated osteocytic cells obtained from bone chip outgrowth cultures (BCOC) to re-differentiate into mature osteoblasts in vitro and in vivo, following a transplantation protocol will be assessed. We will also evaluate the effects of PTH on the osteocyte de-differentiation and changes in osteocyte gene expression utilizing an in vivo model of intermittent treatment with PTH. This will be achieved by utilizing osteocyte specific DMP-1Cre transgenic mice and ZEG (GFP reporter mouse) that would allow also for the lineage tracing of the cells following PTH treatment. Equally important will be to define the changes in expression of genes responsible for matrix removal and de-differentiation process in vivo under PTH influence. Utilizing the transgenic approach and flow cytometry we will selectively isolate the RNA from the osteocytes (DMP1 expressing cells), following an intermittent treatment with PTH. This will allow for the study of the mechanism by which the PTH exerts its effects on this mature osteoblast lineage population and induces matrix removal. PUBLIC HEALTH RELEVANCE: This proposal seeks to evaluate de-differentiation of mature osteoblast lineage cells (preosteocyte/osteocyte) and to establish this process as an integral aspect of bone remodeling. We will evaluate if treatment with parathyroid hormone could induce this de-differentiation process. This would then follow by the evaluation of the effects of various potential therapeutic agents that would be involved in regulation of de-differentiation. The knowledge developed in this grant will provide the foundation for future studies aimed at evaluating factors that would affects process of de-differentiation with the goal of increasing the bone mass in osteoporosis and other disorders with low bone mass.

描述（由申请人提供）：目前，没有证据表明成熟成骨细胞谱系细胞具有去分化的能力。然而，如果可能的话，成熟成骨细胞谱系细胞的去分化可以提供能够增殖和分化的成骨细胞群体的额外来源。我们假设，在成骨细胞谱系活性的高需求的条件下，前骨细胞和骨细胞可以经历去分化，伴随着基因表达的显着变化。这一过程将产生可以表现出增殖潜力的细胞，然后分化成骨基质生产成骨细胞。我们的初步数据支持这些假设，并在此建议中，我们试图确定是否去分化成骨细胞系细胞是骨重建的一个组成部分，并评估相关基因表达的变化。 PTH的间歇给药导致骨合成代谢反应，但所涉及的机制尚未完全了解。我们推测PTH诱导的成骨细胞系去分化可能有助于这种骨合成代谢反应。为了验证这些假设，我们提出了一个在体内评价去分化-再分化的前骨细胞/骨细胞。利用转基因模型，我们可以实现成熟成骨细胞的完全消融，我们将评估去分化的前骨细胞/骨细胞重新填充成骨细胞生态位的能力。此外，将评估从骨芯片生长培养物（BCOC）获得的去分化骨细胞在移植方案后在体外和体内再分化成成熟成骨细胞的能力。 我们还将评估PTH对骨细胞去分化的影响，并利用PTH间歇治疗的体内模型来评估骨细胞基因表达的变化。这将通过利用骨细胞特异性的GFP-1Cre转基因小鼠和ZEG（GFP报告小鼠）来实现，这也将允许在PTH处理后对细胞进行谱系追踪。 同样重要的是确定在PTH影响下负责体内基质去除和去分化过程的基因表达的变化。利用转基因方法和流式细胞术，我们将选择性地从骨细胞（表达DMP 1的细胞）中分离RNA，然后用PTH进行间歇性处理。这将允许研究PTH对该成熟成骨细胞谱系群体发挥其作用并诱导基质去除的机制。 公共卫生相关性：该建议旨在评估成熟成骨细胞谱系细胞（前骨细胞/骨细胞）的去分化，并将此过程作为骨重建的一个组成部分。我们将评估甲状旁腺激素治疗是否能诱导这种去分化过程。随后将评估参与去分化调节的各种潜在治疗剂的作用。在这项赠款中开发的知识将为未来的研究提供基础，旨在评估影响去分化过程的因素，目的是增加骨质疏松症和其他低骨量疾病的骨量。