初级纤毛作为电磁信号感受器调节骨形成的作用与机理研究

The application of electromagnetic fields (EMFs) in the prevention and treatment of osteoporosis has been hot topics for a long time. However, the clinical application of EMFs is still limited today because the development of therapy apparatus has been retarded by poorly understanding of EMF action mechanism. Primary cilium is a solitary, nonmotile microtubule based organelle projecting into extracellular enviroment from cell surface, which has been identified to be cell's antenna for senseing and conveying multiple biochemical and mechanical signals. Here we hypothesized that primary cilia should be ideal sensors for low frequency EMFs and play important roles in regulating the function of bone cells. Basing on the previous works that obtained positive results, the following experiments were designed and would be performed step by step in order to identify our hypothesis. First, the EMF sensor function of primary cilia would be examined by deletion of primary cilia and comparison between the deciliated bone cells and ciliated ones for their osteogenic activity. Second, the effects of EMFs on the morphology and incidence of primary cilia would be investigated by immunofluorescence staining, laser confocal microscopy and scanning electron microscope. The three dimensional cell culture would also be used to avoid the deflection inhibition of primary cilia by common cell culture. Nextly, the regulation of primary cilia in intracellular concentration of second messengers including calcium ion, NO, cGMP and cAMP would be investigated. The control mechanism of intracellular calcium ion by polycystin1/polycystin2 (PC1/PC2) complex and the downstream effectors of NO in primary cilia would be studied by RNA interference, immunofluorescence location and western blot, etc. Fourth, the integrin, a membrane spanning protein coupling the cell to its enviroment known as mechanoreceptor and found to exist in primary cilia, would be studied for its role in transduction of EMF signals by FAK pathway. The in situ proximity ligation assay (in situ PLA) would be used to verify the possible interaction between integrin and PC1. Lastly, Wnt pathway, which play important roles in osteogenic differentiation but was reported to be inhibited by primary cilia, would be evaluated for its activity with report gene assay after treatemnt by EMFs. The effects of inversin, an inhibitor of Wnt pathway released from primary cilia, would be investigated through immunofluorescence location and colocation examination with Dvl and β-catenin. In general, all experiments were designed to demonstrate the EMF sensor function of primary cilia and clarify the mechanism of transducing extracellular EMF signals into intracellular biochemical signals. Besides, the results obtained from the experiments would help to explain the "window effects" of low frequency EMFs in enhancing osteogenic differentiation, which is important in improving the theraputic effect of osteoporosis by EMFs.

电磁场防治骨质疏松症是近年来的研究热点，但因机理不明，导致进展缓慢，临床应用受限。初级纤毛是脊椎动物细胞表面的单根凸起结构，内含丰富的受体、离子通道和激酶等，近年来已被证明是接收多种化学和力学信号的"天线"。我们推测，初级纤毛在胞外低频电磁信号转变为胞内化学信号的过程中扮演着关键角色，是理想的电磁信号感受器和转换器。在前期研究已初获肯定结果的基础上，本项目拟采用免疫荧光染色、激光共聚焦显微镜、三维立体细胞培养、RNA干扰和in situ PLA等技术方法开展以下研究：⑴电磁场对骨细胞系初级纤毛形态与发生率的影响；⑵初级纤毛对胞内钙离子、NO、cGMP和cAMP浓度的调控作用及其机制；⑶整合素进入初级纤毛参与电磁信号转导的机制；⑷初级纤毛对Wnt信号途径的调控与机制。上述研究对深入了解低频电磁场调节骨重建的机理、阐明其促进骨形成"窗效应"的本质及指导提高骨质疏松疗效具有十分重要的意义。

低频电磁场防治骨质疏松症具有无损伤和使用方便等特点，但由于其作用机制不明、疗效不确切而至今无法推广应用。我们在前期已获得“窗效应”电磁场治疗参数的基础上，在国内外首次提出初级纤毛可能是细胞表面的电磁信号感受器，并负责将胞外物理信号转变为胞内化学信号。为证明这一假说，我们采用RNA干扰、免疫荧光染色、激光共聚焦显微镜观察等方法研究了低频电磁场对成骨细胞初级纤毛的影响、初级纤毛被干扰后胞内第二信使、成骨相关信号途径及成骨活性的改变等。结果发现：1.初级纤毛为电磁场促进成骨细胞成骨性分化所必须，没有初级纤毛的成骨细胞对电磁场无成骨反应；2.低频电磁场可以改变初级纤毛的长度和发生率，不同频率、强度、处理时间和波形对初级纤毛的影响不一样，能够延长初级纤毛者不一定具有促进骨形成活性。3.低频电磁场通过激活BMP2/BMPRII/Smad信号途径促进骨形成。初级纤毛根部含有BMPRII，初级纤毛通过控制BMPRII的表达量而控制该信号途径促的活性；4.低频电磁场通过激活AC10/cAMP/PKA信号途径促进骨形成。初级纤毛内含有AC10和p-PKA，干扰初级纤毛可阻止PKA磷酸化。初级纤毛可能通过控制AC10的表达和PKA磷酸化而影响该信号途径的活性；5.低频电磁场通过激活Wnt10b/β-catenin信号途径促进骨形成。Wnt10b可以进出初级纤毛，初级纤毛通过Wnt10b控制该信号途径的活性；6.低频电磁场可以显著减少模拟失重环境的吊尾大鼠发生的骨流失。模拟失重环境使成骨细胞的初级纤毛变短甚至消失，细胞松弛素D可以保护初级纤毛并使成骨细胞保持成骨活性，表明初级纤毛可能是潜在的防治失重引起骨质疏松的靶点。上述研究结果表明，初级纤毛参与了低频电磁场促进骨形成活动，并扮演着不可或缺的重要角色，其作用机制是通过控制多条成骨相关信号途径的关键酶、受体或配体的表达及胞内分布而影响这些信号途径的活性。某些特定电磁场可以显著延长初级纤毛的长度，但这种延长效应不一定就导致成骨活性的增加。本项目研究结果为阐明低频电磁场促进骨形成作用机制开辟了一条新途径。