Fluid Shear Stress and Osteoblast Apoptosis

描述（由申请人提供）：众所周知，机械载荷增加骨的形成和重塑。锻炼会增加骨量，而长期的骨骼卸除，如长时间卧床休息和太空飞行时的微重力，会导致骨骼萎缩。我们研究的主要目的是了解调节机械诱导骨形成的细胞和分子机制。实验研究表明，流体剪切应力（FSS）诱导的成骨细胞活化可能会刺激机械诱导的骨形成。成骨细胞的机械活化被认为是由于在高冲击载荷下，间质液通过骨内多孔空间的运动导致成骨细胞受到FSS的影响。然而，FSS促进成骨细胞合成代谢反应的细胞机制尚不清楚。有趣的是，骨重塑部位的大部分成骨细胞注定要经历程序性细胞死亡（凋亡）。因此，抑制成骨细胞凋亡的过程可能在正常和疾病状态下有效地增加骨形成和提高骨强度。我们的初步研究表明，通过暴露于稳定的流体剪切应力对体外成骨细胞进行机械刺激，可抑制成骨细胞凋亡。因此，在本应用中，我们提出了旨在研究FSS促进成骨细胞存活的信号机制的实验。我们将：(1)确定FSS调节细胞内信号通路参与细胞凋亡控制的机制；(2)确定时间剪切梯度在成骨细胞对FSS的抗凋亡反应中的作用。本研究的长期目标是通过更好地了解调节成骨细胞存活的细胞和分子机制来确定改善骨健康的策略。在本应用中，我们建议使用体外细胞培养模型来验证细胞暴露于稳定或脉动流体剪切应力下通过特定细胞过程调节成骨细胞存活的假设，包括激活pi3 -激酶/Akt和MAPK信号通路以及抑制caspase激活。我们将使用大鼠颅骨成骨细胞和成骨细胞系（包括MC3T3-E1和UMR106.01细胞）的原代培养来研究调节细胞凋亡的细胞机制。

DESCRIPTION (provided by applicant): It is well known that mechanical loading increases formation and remodeling of bone. Bone mass is increased in response to exercise, while chronic unloading of bone, such as occurs during prolonged bed rest and in microgravity during space flight, results in atrophy of bone. The broad aim of our research is to understand the cellular and molecular mechanisms that regulate mechanically-induced bone formation. Experimental studies suggest that mechanically-induced bone formation may be stimulated by fluid shear stress (FSS)-induced activation of osteoblasts. Mechanical activation of osteoblasts is thought to result from the movement of interstitial fluid through the porous spaces inside bone that subjects osteoblasts to FSS during high impact loading. However, the cellular mechanisms through which FSS promotes an anabolic response in osteoblasts are not clearly understood. Interestingly, a large proportion of osteoblasts at sites of bone remodeling are destined to undergo programmed cell death (apoptosis). Therefore, processes that inhibit osteoblast apoptosis may be effective in increasing bone formation and improving bone strength in normal and disease states. Our preliminary studies indicate that mechanical stimulation of osteoblasts in vitro, by exposure to steady fluid shear stress, inhibits osteoblast apoptosis. Therefore, in this application we propose experiments that are designed to investigate the signaling mechanisms through which FSS promotes the survival of osteoblasts. We will: (1) determine the mechanisms through which FSS regulates intracellular signaling pathways involved in control of apoptosis, and (2) determine the role of temporal shear gradients in the anti-apoptotic response of osteoblasts to FSS. The long-term goal of this research is to identify strategies for improving bone health by better understanding the cellular and molecular mechanisms that regulate osteoblast survival. In this application, we propose to use an in vitro cell culture model to test the hypothesis that exposure of cells to either steady or pulsatile fluid shear stress regulates osteoblast survival through specific cellular processes, including activation of the PI3-kinase/Akt and MAPK signaling pathways and inhibition of caspase activation. We will use primary cultures of rat calvarial osteoblasts, and osteoblast cell lines, including MC3T3-E1 and UMR106.01 cells, to investigate the cellular mechanisms that regulate apoptosis.