OSTEOBLASTOGENESIS IN MODELED MICROGRAVITY

描述（由申请人提供）：骨丢失是航天以及老化和废用的主要后果。这些条件的特点是间充质干细胞的成骨细胞生成减少和脂肪生成增加。cbfa 1是成骨细胞分化和骨形成的重要转录因子。此外，PPAR-gamma对脂肪细胞分化也是至关重要的。考虑到骨生成和脂肪生成之间的这种强烈关系，我们假设模拟微重力破坏了整合素介导的信号传导，导致Cbfa 1和PPAR（在人间充质干细胞（hMSC）中）的表达和/或激活发生改变，这导致成骨细胞分化受到抑制并增加脂肪生成。 为了检验这一假设，hMSC将在具有高纵横比容器的旋转细胞培养系统中生长。本研究的目的是：（1）研究模拟微重力对人骨髓间充质干细胞分化的影响。将通过碱性磷酸酶、von Kossa、油红O和三色染色检查成骨细胞生成和脂肪生成的调节。将进行RNA分析以证明成骨细胞和脂肪形成基因表达的调节。(2)描述模拟微重力对人类间充质干细胞影响的分子机制。蛋白质印迹和凝胶迁移分析和反式激活研究将被用来阐明机制。(3)鉴定能够逆转成骨细胞生成的模拟微重力抑制的药物。使用与目标1和2相同的技术，我们将鉴定能够阻断微重力诱导的骨丢失的药物。这些研究的结果将深入了解失重对间充质细胞功能的影响所涉及的机制，从而满足本PA的要求（PA-00-088）。因此，将为开发能够改变Cbfa 1和/或PPARgamma并介导宇航员、老年人和废用性骨质疏松症患者骨形成增加的药物提供可行的基础。

DESCRIPTION (provided by applicant): Bone loss is a major consequence of spaceflight as well as aging and disuse. These conditions are marked by a decrease in the osteoblastogenesis of mesenchymal stem cells and an increase in adipogenesis. Cbfa1 is an essential transcription factor in osteoblast differentiation and bone formation. In addition, PPAR-gamma was shown to be crucial for adipocyte differentiation. Given this strong relationship between osteogenesis and adipogenesis, we hypothesize that modeled microgravity disrupts integrin-mediated signaling leading to alterations in the expression and/or activation of both Cbfa1 and PPAR( in human mesenchymal stem cells (hMSC), which results in suppressed osteoblast differentiation and increase adipogenesis. To test this hypothesis, hMSC will be grown in a Rotary Cell Culture System with a high aspect ratio vessel. The aims of this study are: (1) Characterize the effects of modeled microgravity on human mesenchymal stem cell differentiation. Modulation of osteoblastogenesis and adipogenesis will be examined by alkaline phosphatase, von Kossa, oil red O and trichrome staining. RNA analyses will be performed to demonstrate the modulation of osteoblastic and adipogenic gene expression. (2) Characterize the molecular mechanisms responsible for the effects of modeled microgravity on human mesenchymal stem cells. Western blot and gel shift analysis and transactivation studies will be utilized to elucidate the mechanism. (3) Identifying agents capable of reversing the modeled microgravity inhibition of osteoblastogenesis. Using the same techniques employed in Aims 1 and 2, we will identify agent/s capable of blocking microgravity-induced bone loss. Findings from these studies will provide insight into mechanisms involved in the effect of weightlessness on mesenchymal cell function, thus addressing the requirements of this PA (PA-00-088). A viable basis will thereby be provided for the development of drugs that can alter Cbfa1 and/or PPARgamma and mediate increased bone formation in astronauts, elderly and patients with disuse osteoporosis.