Ca2+/NFAT信号通路在成骨细胞脉冲电磁信号转导中的作用及机制研究

Pulsed electromagnetic fields (PEMF) are capable of promoting bone fracture healing and inhibiting bone loss. However, the potential mechanism by which PEMF stimulation affects the skeleton remains unclear thus far, which greatly limits the extensive and scientific application of PEMF in clinics. The findings from our group demonstrate that osteoblasts exhibit unique cytosolic Ca2+ oscillation with multiple Ca2+ spikes in response to PEMF stimulation, revealing that osteoblasts as the key target cells sensing external electromagnetic signals in the skeleton can respond to exogenous PEMF stimulation via unique Ca2+ oscillation. However, the mechanism by which osteoblasts decode and transduce this unique Ca2+ oscillation remain unclear. Substantial evidence has shown that NFAT exhibits the decoding effects for low-frequency Ca2+ oscillation in kidney cells and cardiac cells. Thus, the current project aims to analyze the spatiotemporal distribution characteristics of intracellular Ca2+ and NFAT in osteoblasts subjected to PEMF stimulation via dual-channel Ca2+ and NFAT confocal imaging and thereby clarifies the decoding effects of NFAT on PEMF-induced Ca2+ oscillation in osteoblasts. Then, we will also analyze osteoblast activities and functions in response to PEMF stimulation after the NFAT signaling is blocked, and thus clarify the transducing effects of NFAT on PEMF-induced Ca2+ oscillation in osteoblasts. Together, the present investigation aims to systematically clarifies the mechanism by which osteoblasts decode and transduce the calcium signaling under the PEMF stimulation, which is helpful for advancing our understanding for the mechanism about bone’s electromagnetic signal transduction and how PEMF promote bone formation and fracture repair, and providing important experimental evidence for more scientific clinical application of PEMF in the treatment of bone diseases.

脉冲电磁场（PEMF）能够加速骨折愈合并抑制骨丢失，但是它对骨骼的作用机制尚未阐明，这也限制了其广泛科学的临床应用。我们的实验表明PEMF能诱发成骨细胞（OB）产生胞浆“多峰钙振荡”这一独特现象，揭示了OB作为骨感应PEMF的主要靶细胞通过钙振荡来感应处理外源性电磁信号，但是OB对于这种钙振荡的解析和转导机制仍不明确。研究发现NFAT在心脏、肾脏等细胞中对低频率钙振荡具有解码效应。本项目拟采用NFAT与Ca2+双通道共聚焦成像研究PEMF刺激下OB胞浆NFAT与Ca2+时间-空间分布特征，阐明NFAT在PEMF诱发的OB钙振荡的解码功能；随后研究NFAT受抑制的OB在PEMF刺激下生物活性功能的改变，明确NFAT在OB钙信号转导中的作用。本项目旨在阐明电磁作用下OB钙信号的解析和转导机制，从而深化对于骨的电磁信号转导及PEMF促成骨机理的认识，为PEMF更科学的临床骨疾病治疗提供重要依据。

本课题从PEMF激活OB中钙/NFAT信号这一崭新角度为切入点，系统探索了OB对于PEMF刺激的信号响应和转导机制，主要内容包括：（1）研制了基于3-Helmholz线圈的新型程控多波形电磁发生系统、多尺度的骨及骨细胞机械应力加载系统，该系统所产生的电磁场空间分布均匀性显著高于传统的2-Helmholz线圈系统。（2）构建了胰岛素依赖的速发型糖尿病诱导的骨质疏松模型和糖皮质激素地塞米松诱导的骨质疏松模型，并在股骨的外侧髁构建骨缺损，植入我们前期研制的低弹多孔钛合金，发现PEMF刺激能够显著改善两种骨质疏松兔的小梁骨和皮质骨的骨量和微观结构，显著提高皮质骨和小梁骨的结构和材料力学属性，显著促进多孔钛合金植入体在骨缺损位置的骨整合能力，而这与PEMF对于骨中OB介导的骨形成速率的促进作用相关。（3）通过使用新颖的微接触压印和自组装单分子表面化学技术成功构建了体外OB的拓扑网络，发现PEMF刺激下OB表现出独特的多尖峰钙振荡特性，揭示了OB主要通过胞浆多尖峰钙振荡响应外界的电磁信号刺激。（4）筛选明确了OB胞内NFATc3亚型，通过构建钙-NFAT双通道荧光实时成像技术，发现了PEMF刺激下OB胞内NFATc3活性与细胞钙振荡呈现正相关关系，揭示了NFAT充当着OB响应外界PEMF刺激下胞内多尖峰钙振荡解码器的角色。（5）通过使用基因沉默技术阻断OB中的NFATc3表达，同时构建OB特异性NFATc3敲除小鼠，发现当NFATc3受抑制后，PEMF所诱发的OB的细胞增殖、分化、矿化以及成骨因子表达的增加被显著抑制，同时OB中NFATc3条件性敲除小鼠的骨量、骨微结构、骨矿化沉积率等也显著抑制，而PEMF刺激无法显著逆转这种抑制效应，进一步揭示了NFAT不仅充当着PEMF刺激下OB钙振荡的重要“解码器”的角色，同时对于PEMF的钙振荡的下游信号“转导”同样发挥重要作用。