OSTEOBLASTOGENESIS IN MODELED MICROGRAVITY

描述(申请人提供)：骨质流失是航天飞行以及老化和废弃的主要后果。这些情况的显著特征是间充质干细胞的成骨细胞生成减少，脂肪生成增加。Cbfa1是成骨细胞分化和骨形成过程中必不可少的转录因子。此外，PPAR-γ被证明是脂肪细胞分化的关键。鉴于成骨和脂肪生成之间的这种密切关系，我们假设微重力模型干扰了整合素介导的信号转导，导致Cbfa1和/或PPAR(在人骨髓间充质干细胞(HMSC)中的表达和/或激活)的变化，从而导致成骨细胞分化受抑，脂肪生成增加。 为了验证这一假设，hMSC将在具有高长宽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。碱性磷酸酶、von Kossa、油红O和三色染色将检测成骨细胞和脂肪生成的调控。将进行RNA分析，以证明成骨和成脂基因表达的调节。(2)研究模拟微重力对人骨髓间充质干细胞作用的分子机制。将利用蛋白质印迹和凝胶位移分析以及反式激活研究来阐明其机制。(3)确定能够逆转模拟的微重力对成骨细胞生成的抑制的药物。使用AIMS 1和AIMS 2中使用的相同技术，我们将确定能够阻止微重力引起的骨丢失的制剂/S。这些研究的结果将有助于深入了解失重对间充质细胞功能的影响机制，从而满足本PA的要求(PA-00-088)。因此，将为开发能够改变Cbfa1和/或PPARγ并调节宇航员、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.