Osteocytes and Mechano-Transduction

描述(申请人提供)：骨细胞是骨骼中最丰富的细胞，被认为在骨骼机械传感中起着关键作用，它们通过调节骨骼建模和重塑来响应剪切力或应变的变化。尽管众所周知，骨骼对其机械环境做出反应，但机械转导途径背后的机制却知之甚少。此外，最近的发现证明了骨细胞在调节磷酸盐稳态中的作用。使用机械活动或静态培养条件培养骨细胞，我们将验证SOST和成纤维细胞生长因子-23是机械感觉的重要调节因素的假设。初步研究将致力于建立有条件永生化的骨细胞系，该细胞系来源于在DMP-1(已知仅在骨细胞中表达)控制下表达荧光绿色蛋白(GFP)的转基因小鼠，并携带温度敏感的永生化SV40抗原。细胞将在3D支架上生长，并受到1g、微重力(NASA水平生物反应器)或增加机械刺激(NASA垂直生物反应器)。然后，我们将通过使用小干扰RNA(SiRNA)选择性地沉默这些转录本，来研究SOST和FGF23信号在骨细胞中的作用(在静态和动态条件下)。分析在这些条件下的基因表达模式，并测量硬化素和FGF23的分泌，将为深入了解机械转导途径和矿质离子调节(UH2)提供线索。最后，利用国际空间站(ISS)提供的微重力环境和最小流体剪切培养条件，我们建议研究骨细胞对卸载条件(UH3)的反应。EOSTEO飞行硬件将根据这次国际空间站任务的目标进行相应的修改。该提案的执行伙伴是CAMM技术公司和加拿大航天局(CSA)。所提出的研究结果可能对与停用或制动相关的骨病的治疗具有重要意义。

DESCRIPTION (provided by applicant): Osteocytes, the most abundant cells in bone, are believed to play a key role in skeletal mechanosensing whereby they modulate bone modeling and remodeling in response to changes in shear or strain forces. Although it is well-established that bone responds to its mechanical environment, the mechanisms underlying the mechano- transduction pathway are poorly understood. Moreover, recent findings have documented a role of osteocytes in the regulation of phosphate homeostasis. Using a mechanically active or static culture condition to culture osteocytes, we will test the hypothesis that SOST and FGF-23 are important regulators of mechano-sensation. Initial studies will be devoted to establish conditionally immortalized osteocytic cell lines derived from transgenic mice expressing the fluorescent green protein (GFP) under the control of DMP-1 (known to be expressed exclusively in osteocytes) and carrying the temperature-sensitive immortalizing SV40 antigen. Cells will be grown on 3D scaffold and subjected to 1g, microgravity (NASA horizontal bioreactor) or increase mechanical stimulation (NASA vertical Bioreactor). We will then investigate the role of SOST and FGF23 signaling in osteocytes (under static and dynamic condition) by selectively silencing these transcripts using small interfering RNAs (siRNA). Analysis of gene expression patterns under these conditions and measuring secretion of sclerostin and FGF23 will provide insights into mechano-transduction pathways and mineral ion regulation (UH2). Lastly utilizing the microgravity environment and minimal fluid shear culture conditions available only onboard of the International Space Station (ISS) we propose to investigate osteocytes responses to unloading conditions (UH3). The eOSTEO flight hardware will be modified accordingly to the aims of this ISS mission. Implementation Partner for this proposal are Calm Technologies and the Canadian Space Agency (CSA). Results derived from the studies proposed could have significant implications for therapy of bone disorders related to disuse or immobilization.