Evaluation of Osteocyte Dedifferentiation Process

描述(由申请人提供)：目前，没有证据表明成熟的成骨细胞系细胞具有去分化的能力。然而，如果可能，成熟的成骨细胞系细胞的去分化可以提供一个额外的成骨细胞群体的来源，能够增殖和分化。我们假设，在对成骨细胞系活性要求很高的条件下，前骨细胞和骨细胞可以经历去分化，并伴随着基因表达的急剧变化。这一过程将产生具有增殖能力的细胞，然后分化为骨基质，产生成骨细胞。我们的初步数据支持这些假设，在这个建议中，我们试图确定成骨细胞系细胞的去分化是否是骨重建的一个组成部分，并评估涉及的基因表达的变化。间歇性应用甲状旁腺素可引起骨合成代谢反应，但其机制尚不完全清楚。我们推测，甲状旁腺素诱导的成骨细胞系细胞的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，我们提出了一项对前骨细胞/骨细胞去分化-再分化的体内评估。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，还将评估从骨片生长培养(BCOC)中获得的去分化骨细胞在体外和体内按照移植方案重新分化为成熟成骨细胞的能力。我们还将利用甲状旁腺素间歇处理的体内模型来评估甲状旁腺素对骨细胞去分化和骨细胞基因表达变化的影响。这将通过利用骨细胞特异性DMP-1Cre转基因小鼠和ZEG(GFP报告小鼠)来实现，这也将允许对PTH治疗后的细胞进行谱系追踪。同样重要的是确定在甲状旁腺激素影响下体内负责基质去除和去分化过程的基因的表达变化。利用转基因方法和流式细胞术，我们将有选择地从骨细胞(表达DMP1的细胞)中分离出RNA，然后用甲状旁腺素间歇处理。这将有助于研究甲状旁腺激素对这个成熟的成骨细胞系群体发挥作用并诱导基质去除的机制。 公共卫生相关性：这项建议旨在评估成熟成骨细胞系细胞(前成骨细胞/骨细胞)的去分化，并将这一过程确立为骨重建的一个不可或缺的方面。我们将评估甲状旁腺激素治疗是否可以诱导这种去分化过程。随后将对参与调节去分化的各种潜在治疗剂的效果进行评估。这笔赠款中发展的知识将为未来旨在评估影响去分化过程的因素的研究提供基础，目的是增加骨质疏松症和其他骨量低的疾病的骨量。

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.