Cav1.2在电磁场调控骨形成改善老年性骨质疏松症中的作用及机制研究

The incidence of osteoporosis (OP) has increased year by year with the aging of China's population. Existing drug therapies require long-term medication, and their safety is worrying. Finding safe and effective alternative therapies is an urgent problem to be solved. Numerous studies have shown that electromagnetic fields (EMF) can promote bone formation and improve OP, but its regulation mechanism is unclear. The previous study of the research team found that the activation of L-type voltage-gated Ca2+ channel (VGCC) in osteoblasts is closely related to EMF the promoting of bone formation by EMF. However, there are four types of VGCC. In the end, which VGCC is involved in regulating EMF to promote bone formation remains to be explored. It was further found in the preliminary experiment that EMF with specific frequency can significantly improve the bone formation reduction caused by blocking Cav1.2 which is one type of VGCC. Therefore, the research team proposed a scientific hypothesis that the activation of Cav1.2 L-type VGCC by EMF increases Ca2+ influx, as a result of promoting bone formation and improving OP. This project intends to reveal the role and mechanism of Cav1.2 in promoting bone formation in EMF by comprehensively calculating the electromagnetic characteristics of Cav1.2, Ca2+ fluorescent probe technology, the patch clamp technique and molecular biology technology. In this way, the role and mechanism of Cav1.2 in promoting bone formation in EMF were revealed, which provided a target and theoretical basis for clinical application of EMF to promote bone formation and improve OP.

骨质疏松症（OP）的发病率随着我国人口老龄化的持续加深逐年攀升。现有的药物疗法需长期服药，其安全性令人担忧，寻找安全有效的替代疗法是目前亟待解决的问题。大量研究表明电磁场（EMF）能促进骨形成，改善OP，但其调控机制不清。课题组前期研究发现，成骨细胞L型电压门控Ca2+通道（VGCC）的激活与EMF促骨形成密切相关。但是VGCC包含四种，到底哪种VGCC参与调控EMF促进骨形成，这一问题有待探索。预实验中进一步发现，EMF可显著改善阻断Cav1.2这一类型VGCC造成的骨形成降低。由此，课题组提出EMF激活Cav1.2 L型VGCC增加Ca2+内流是促进骨形成改善OP的重要机制的科学假说。本课题拟综合工学建模计算Cav1.2电磁特性、Ca2+荧光探针技术、膜片钳技术以及分子生物学技术等方法，揭示Cav1.2在EMF促骨形成中的作用和机制，为EMF促骨形成改善OP临床应用提供靶点和理论依据。

本项目围绕“特定频点电磁场（EMF）激活Cav1.2 L 型电压门控钙离子通道（VGCC）增加Ca2+内流是促进骨形成改善骨质疏松（OP）的重要机制”这一科学假说，综合工学建模计算及分子生物学实验手段，旨在揭示Cav1.2在EMF促进骨形成的作用和机制。.研究结果发现：（1）Cav1.2三维结构模型的构建和对Cav1.2固有电磁特性量化计算的结果显示能够引起该通道蛋白上精氨酸和赖氨酸转动的电磁场频段在GHz频段，因而从经典物理学角度无法解释大量文献报道的低频电磁场（≤300Hz）能够促进成骨细胞增殖和分化这一现象。本研究根据量子共振理论，认为频率为38.7Hz的电磁场可引发大量Ca2+协同化的同向运动，激活了电压敏感性钙离子通道，导致细胞内Ca2+增多进而产生系列生物学效应。因而，提出的“特定频段EMF可能激活Cav1.2 L 型VGCC是EMF促进骨形成，是改善OP的可能机制”这一科学假说可能是潜在的电磁促进骨形成的可能机理。（2）分子生物学实验结果显示，频率为38.7Hz，强度为1.4mT（有效值）的正弦电磁场，能够促进MC3T3-E1成骨细胞的增殖、分化，能够促进成骨细胞内Ca2+内流，导致Ca2+相关信号通路介导了成骨细胞的增殖和分化，起到促成骨的生物效应。（3）分子生物学实验结果尽管显示EMF干预会导致成骨细胞内Ca2+增多，但是对于Cav1.2这一电压敏感的钙离子膜通道蛋，不论是从基因还是蛋白的角度对其量的表达均没有影响，可能的原因在于EMF仅对其功能起调控作用。（4）为此，我们提出了一种科学假说：Cav1.2离子通道可以看作“生命系统中的三极管”，Cav1.2位于成骨细胞膜外侧的亚基可以看成三极管的基极，Cav1.2与细胞膜相接触的亚基可以看成三极管的源极，Cav1.2位于成骨细胞膜内侧的亚基可以看成三极管的集电极。这样，细胞膜外侧低频电磁场引起的少量Ca2+的共振，可以引起成骨细胞膜里大量Ca2+内流，进而可以引起促成骨的生物效应。.本项目探究了Cav1.2在电磁场影响成骨细胞增殖与分化中的作用及机制，为揭示EMF对骨的非热生物效应作用机理提供了新的思路与实验依据，为EMF促进骨形成改善OP临床应用提供靶点和理论依据。