Regulatory factors on osteoclast formation and function.

破骨细胞形成和功能的调节因素。

• 批准号: 1905937
• 金额: --
• 依托单位: Royal Veterinary College
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1905937
• 关键词: Regulatory; factors; osteoclast; formation; function.

It is increasingly recognised that exosomes can contribute to the pathogenesis of bone disease. This collaborative research project will investigate how exosomes regulate bone remodelling under normal and stress conditions. As such the proposal fits well with the BBSRC research priority 3, Bioscience for Health, and in particular the areas of "healthy ageing across the life course" and "the 3Rs in research using animals".Bone homeostasis reflects the tightly regulated actions of osteocytes (OCY), osteoblasts (OB) and osteoclasts (OC); dysregulation of these systems can lead to bone disease. OB are the bone-forming cells which, when incorporated into bone matrix, can terminally differentiate into OCY. OCY, the most abundant cells in bone, form an extensive communication network that plays a central role in regulating bone turnover. O2 tension and pH have a profound effect on bone cell function with hypoxia and acidosis being implicated in the development of diseases such as osteoporosis. We have shown that hypoxia and acidosis stimulate OC formation and resorptive activity whilst inhibiting bone mineralisation. Moreover, acute hypoxia stimulates vesicular ATP release from OB.Exosomes are small membrane-bound particles released from cells. They contain a diverse array of signalling molecules (e.g. proteins, miRNAs) and are increasingly viewed as key mechanism by which cells regulate local extracellular signalling. Evidence from different cell types suggests that environmental stress factors like hypoxia have a profound effect on exosome composition and release.This studentship will investigate the hypothesis that "environmental stress affects bone cell function by altering exosome release and composition". The research objectives are:1. Defining the role of OB and OCY-derived exosomes in the regulation of bone cell function The first objective of this studentship will be to fully characterise the composition of OB and OCY-derived exosomes under normal conditions. The ability of isolated exosomes to regulate bone cell differentiation, function (i.e. bone formation/resorption) and gene expression will also be examined. This in vitro project will use primary OB and OC along with IDG-SW3 cells (an OB-to-late-OCY cell line). Experimental approaches that will be used to address the hypothesis include:- Transmission electron, confocal and real-time fluorescence microscopy to examine exosome release and uptake- FACS characterisation of isolated exosomes- RNAseq and bioinformatics analysis of vesicle contents - Real-time PCR, western blotting and/or ELISA for studying genes of interest- Established assays for measuring bone formation and resorptionBone cell co-cultures will also be used to establish if the actions on cell, differentiation and function recapitulate what is seen with isolated exosomes. Using these co-culture systems places the project at the cutting edge of in vitro bone biology techniques. Moreover, they allow bone formation and resorption to be studied without the need for in vivo work.2. Understanding the effects of hypoxia and acidosis on OB and OCY-derived exosomes The effect of long-term and acute exposure to hypoxia (2% O2) or acidosis (pH6.9) on exosome-mediated signalling in bone will be studied using the experimental approaches described in objective 1. This work will give valuable insights into how OB and OCY-derived signals are dysregulated under stress conditions.

越来越多的人认识到，外泌体可能有助于骨疾病的发病机制。这个合作研究项目将研究外泌体如何在正常和应激条件下调节骨重建。因此，该提案非常符合BBSRC研究优先事项3，健康生物科学，特别是“整个生命过程中的健康老龄化”和“使用动物研究中的3R”领域。骨稳态反映了骨细胞（OCY），成骨细胞（OB）和破骨细胞（OC）的严格调节作用;这些系统的失调可能导致骨骼疾病。成骨细胞是骨形成细胞，当其与骨基质结合时，可终末分化为OCY。OCY是骨中最丰富的细胞，形成了广泛的通讯网络，在调节骨转换中起着核心作用。O2张力和pH值对骨细胞功能有深远的影响，缺氧和酸中毒与骨质疏松症等疾病的发展有关。我们已经表明，缺氧和酸中毒刺激OC的形成和吸收活性，同时抑制骨矿化。此外，急性缺氧刺激泡状ATP从OB释放。外泌体是从细胞释放的小的膜结合颗粒。它们包含多种信号分子（例如蛋白质，miRNA），并且越来越多地被视为细胞调节局部细胞外信号的关键机制。来自不同细胞类型的证据表明，环境应激因素（如缺氧）对外泌体的组成和释放有深远的影响。本研究将探讨“环境应激通过改变外泌体的释放和组成来影响骨细胞功能”的假设。本研究的目的是：1.定义OB和OCY衍生的外泌体在调节骨细胞功能中的作用本研究的第一个目标是在正常条件下充分鉴定OB和OCY衍生的外泌体的组成。还将检查分离的外泌体调节骨细胞分化、功能（即骨形成/再吸收）和基因表达的能力。该体外项目将使用原代OB和OC沿着IDG-SW 3细胞（OB-晚期-OCY细胞系）。将用于解决该假设的实验方法包括：-透射电子、共聚焦和实时荧光显微镜检查外泌体释放和摄取-分离的外泌体的FACS表征-囊泡内容物的RNAseq和生物信息学分析-用于研究感兴趣的基因的实时PCR、蛋白质印迹和/或ELISA-用于测量骨形成和吸收的已建立的测定法骨细胞共培养物也将用于确定对细胞、分化和功能的作用是否重演分离的外泌体所看到的。使用这些共培养系统将该项目置于体外骨生物学技术的前沿。此外，它们允许骨形成和吸收进行研究，而不需要在体内工作。了解缺氧和酸中毒对OB和OCY来源的外泌体的影响将使用目标1中描述的实验方法研究长期和急性暴露于缺氧（2%O2）或酸中毒（pH 6.9）对骨中外泌体介导的信号传导的影响。这项工作将提供有价值的见解OB和OCY衍生的信号是如何在应激条件下失调。