Gravitational Regulation of Osteoblast Genomics and Metabolism

描述(申请人提供)：重力在地球上骨骼的发育和维持中的作用是有争议的。我们目前的理解是，骨的重力卸载会导致骨丢失(骨量减少)，这通常会进展为骨质疏松，最终导致骨折。这是卧床不起的患者和老年人遇到的一个重大问题。在轨道上的宇航员每月经历大约1%到3%的骨丢失的严重骨量减少，这相当于地球上骨质疏松症患者一年的骨丢失。了解骨骼对不同水平的重力的反应对于在药物设计或物理治疗中开发合理的方法来缓解骨量减少至关重要。目前，将骨骼暴露在长期缺乏重力的情况下的唯一方法是通过在国际空间站上的轨道自由落体。这些研究费用昂贵，在国际空间站上进行后勤工作也很困难。研究骨骼动力学的重力调节的理想方法是有一种基于地面的方法来消除或减少对骨骼的引力。创造一个扩大骨丢失速度的环境将给我们提供一个工具，以加速我们对潜在的基因组、蛋白质和代谢机制的理解。这项拟议的研究试图确定地面磁悬浮是否适合用于细胞培养研究的轨道自由落体模拟。研究细胞培养而不是整个动物可以进行高度可控和可重复性的实验。我们计划在与地面悬浮磁铁和国际空间站空间微重力环境兼容的生物反应器中培养MC3T3成骨细胞和原代破骨细胞。我们独特的悬浮磁铁集成了一个37摄氏度的孵化器，集成在磁铁孔中，允许悬浮数周。国际空间站上实验的实施伙伴是位于科罗拉多州博尔德市的BioServe空间技术公司。BioServe在与NASA合作将生物实验运送到近地轨道和从近地轨道运送生物实验方面，已经有20年的成就。在之前的轨道飞行任务中飞行的成骨细胞和破骨细胞表明，微重力影响细胞分化和基因表达。这项研究的指标包括细胞形态研究，基因差异表达的微阵列分析，鉴定成骨细胞和破骨细胞分化标志物的ELISA和RT-PCR，代谢组学，通过H-1核磁共振波谱和MAPK，Twsg1和NF-kB信号通路的定量。通过这套分析，我们将确定模拟微重力和国际空间站微重力环境中一些代谢物和生物标记物的异同。这个项目将帮助确定可以解决的减轻地球上的骨丢失的机制(S)和途径。

DESCRIPTION (provided by applicant): The role of gravity in the development and maintenance of bone on earth is controversial. Our current understanding is that gravitational unloading of bone results in bone loss (osteopenia) which typically progresses to osteoporosis and ultimately bone fracture. This is a significant problem that bed- ridden patients and the elderly experience. Astronauts in orbit experience significant osteopenia of approximately 1% to 3% of bone loss per month, which is equivalent to a year of bone loss for osteoporotic individuals on earth. Understanding bone response to varying levels of gravitational force is critical toward developing rational approaches in drug design or physical therapy toward mitigating osteopenia. Presently, the only means of exposing bone to a long-term lack of gravitational force is through orbital free fall aboard the ISS. These studies are expensive and logistically difficult to do aboard the ISS. An ideal method of study gravitational regulation of bone dynamics is to have a ground-based method to remove or reduce the gravitational force on bone. Creating an environment that amplifies the rate of bone loss will give us a tool to accelerate our understanding of the underlying genomic, proteomic and metabolomic mechanisms. The proposed study seeks to determine if ground-based magnetic levitation is a suitable simulation of orbital free fall for cell culture studies. Studying a cell culture rather than an entire animal allows for highly controlled and reproducible experiments. We plan to grow MC3T3 osteoblastic and primary osteoclastic cells in a bioreactor compatible with our ground-based levitation magnet and the ISS space-based microgravity environment. Our unique levitation magnet integrates a 37 C incubator integrated into the magnet bore allowing levitation for several weeks. The implementation partner for experiments aboard the ISS is BioServe Space Technologies in Boulder, CO. BioServe has a two-decade record of accomplishment of working with NASA on delivering biological experiments to and from low-earth orbit. Osteoblasts and osteoclasts flown on previous orbital missions show evidence that microgravity affects cell differentiation and gene expression. The metrics for the proposed study include cell morphology studies, microarray analysis to measure differential gene expression, ELISA and RT-PCR to identify markers of osteoblast and osteoclast differentiation, metabolomics, via H-1 NMR spectroscopy and quantification of MAPK, Twsg1 and NF-kB signaling pathways. With this suite of assays, we will determine similarities and differences in a number of metabolites and biomarkers for simulated microgravity and ISS microgravity environments. This project will help identify mechanism(s) and pathways that can be addressed to mitigate bone loss on earth.