Nucleotides/fluid flow effects on osteoblastic cells

描述(申请人提供)：众所周知，由机械负荷引起的细胞外液流动促进了成骨细胞的代谢、分化、细胞外基质蛋白的产生和基因表达。近年来，越来越多的证据表明细胞外核苷酸在骨细胞的增殖和分化中起着重要的作用。因此，这个项目的中心目标是描述成骨细胞检测细胞外液流动并将其转化为生化反应的机制。我们的中心假设是，生物物理信号，如液体流动，通过涉及胞内钙动员的机制刺激成骨细胞分化，重要的是，这需要细胞外核苷酸的存在。我们将使用荧光成像、生物工程和分子生物学技术的组合来验证这一假设，以完成四个特定的目标。在特定的目标1中，我们将研究细胞外核苷酸是否是成骨细胞内细胞内钙的流动动员所必需的。在特定的目标2和3中，我们将确定特定的P2Y嘌呤能受体在液体流动诱导的细胞内钙浓度增加中的作用。在最终的特定目标中，我们将研究细胞外核苷酸及其特定受体(在特定目标1至3中确定)在流动诱导成骨细胞分化中的作用。这将通过量化三种成骨细胞分化标志物碱性磷酸酶、骨桥蛋白和骨钙素的流动诱导效应来实现。通过完成这些特定的目标，我们将深入了解生物物理信号影响骨细胞行为的机制。这些信息可能会为开发治疗骨量减少症的新疗法提供指导

DESCRIPTION (provided by applicant): It is well known that extracellular fluid flow-induced by mechanical loading increases osteoblastic cell metabolism, differentiation, extracellular matrix protein production and gene expression. Recently, there is increasing evidence suggesting that extracellular nucleotides play an important role in bone cell proliferation and differentiation. Thus, the central goal of this project is to characterize the mechanism by which osteoblastic cells detect extracellular fluid flow and transduce this into biochemical responses. Our central hypothesis is that biophysical signals, such as fluid flow, stimulate osteoblastic cell differentiation via a mechanism involving cytosolic calcium mobilization and, importantly, this requires the presence of extracellular nucleotides. We will test this hypothesis using a combination of fluorescent imaging, bioengineering and molecular biological techniques to complete four specific aims. In specific aim 1 we will examine whether extracellular nucleotides are necessary for fluid flow mobilization of cytosolic calcium in osteoblastic cells. In specific aims 2 and 3 we will determine the roles of specific P2Y purinergic receptors in fluid flow-induced increases in intracellular calcium concentration. In the final specific aim we will examine the effects of extracellular nucleotides and their specific receptors (identified in specific aims 1 to 3) on flow-induced osteoblastic differentiation. This will be achieved by quantifying flow-induced effects on three osteoblastic differentiation markers, alkaline phosphatase, osteopontin, and osteocalcin. By completing these specific aims we will gain insights into the mechanism by which biophysical signals affect bone cell behavior. Such information may provide guidance in developing novel treatments for osteopenia