酸敏感离子通道在整合素αvβ3介导的破骨细胞粘附及骨破坏作用研究

The integrin αvβ3 is an important protein in the cell membrane of osteoclasts, regulates osteoclast adhesion and bone resorption. Recent studies found that the extracellular acidification accelerates bone resorption by enhancing osteoclast adhesion and activation, which is another important cause of osteoporosis (OP). In this study, the applicant found the mechanisms based on the National Natural Science Foundation (Study on the mechanisms of the osteoclast proliferation and activation mediated by acid-sensing ion channels in osteoporosis rats 30,901,526) . we found that Acid-sensing ion channel is involved in acid-induced osteoclast proliferation and activation, providing a means to sense and respond to differences in extracellular pH. In addtion, the activation of ASIC1by acid can significantly promote osteoclast αvβ3 expression. However, the mechanisms by which ASIC1 promote osteoclast αvβ3 expression and bone resorption remain largely unidentified.Thus, the mechanisms needs further study. We will study the role of ASIC1 by ASIC1 selective agonist, antagonist, ASIC1 overexpression, ASIC1 RNA interference and protein microarray in the cell model. The same time, we also further validate superior mechanisms by the model of osteoporosis. The aim of the present study was to establish the expression of ASIC1a in osteoclasts and to identify its function in regulating osteoclast adhesion and bone resorption via integrin αVβ3. So, it has very important theoretical significance and practical value for us to undestand more of the function of ASIC1 and screen new anti-osteoporosis target.

整合素αvβ3是破骨细胞膜上的蛋白，影响破骨细胞粘附及骨吸收能力。近年来发现酸化作用于破骨细胞，增强活化和粘附是发生骨质疏松的又一个重要原因，本研究申请者在上一个国家自然科学基金（酸敏感离子通道在骨质疏松大鼠破骨细胞增殖与活化中的作用机制研究）项目的工作基础上发现，酸敏感离子通道1（ASIC1）介导酸诱导破骨细胞增殖与活化，是破骨细胞膜上的酸受体。此外，酸激活ASIC1能显著促进破骨细胞αvβ3表达。但关于ASIC1促进αvβ3表达后影响破骨细胞粘附和骨吸收作用机制，尚需进一步研究。拟在细胞中通过ASIC1激动剂、拮抗剂、ASIC1过表达转染和RNA干扰以及蛋白芯片等方法，分析其通过αvβ3调控破骨细胞的粘附和骨吸收作用及其信号通路，同时，通过骨质疏松模型进一步验证，意在阐明ASIC1通过αvβ3促进破骨细胞粘附作用机制，对人们更好地认识ASIC1功能及寻找新的抗骨质疏松靶点有重要意义。

研究已经证实细胞外酸化是破骨细胞增殖活化的重要原因之一，我们前期研究发现酸敏感离子通道ASIC1a是酸诱导的破骨细胞增殖活化的酸受体。然而ASIC1a是否介导破骨细胞黏附与迁移有待于进一步研究。我们前期（1）通过siRNA技术敲除破骨细胞中的ASIC1a，探讨其介导破骨细胞骨吸收作用（2）细胞外酸化诱导破骨细胞前体细胞（BMM）和成熟的破骨细胞的迁移和黏附实验，（3）荧光显微镜观察破骨细胞骨架F-actin形成，（4）整合素α vβ 3 在破骨细胞膜上的改变，（5）α vβ 3 调控破骨细胞的粘附和骨吸收作用及其信号通路分析其机制作用。我们研究发现（1）ASIC1a介导破骨细胞BMM和成熟的破骨细胞的黏附和迁移作用，（2）阻断ASIC1a 抑制破骨细胞表面 β3 蛋白的表达及整合素α vβ 3 的改变，（3）阻断ASIC1a抑制破骨细胞骨架F-actin形成，（4）阻断ASIC1a抑制破骨细胞钙离子Ca2+依赖的黏附信号下游分子Pyk2 和 Src的磷酸化，（5）阻断ASIC1a抑制破骨细胞钙离子Ca2+依赖的黏附信号下游分子Pyk2 和 Src蛋白相互作用，（6）阻断ASIC1a抑制破骨细胞ASIC1a和αvβ3 蛋白相互作用。意在阐明 ASIC1 通过α vβ 3 促进破骨细胞粘附作用机制，对人们更好地认识 ASIC1功能及寻找新的抗骨质疏松靶点有重要意义。项目总共发表SCI论文7篇。