嘌呤受体P2X7在低频电磁场促成骨中的作用及机制研究

Postmenopausal osteoporosis (PMOP) and associated fractures are becoming a serious public health issue. Clinical trials show that electromagnetic fields (EMFs) can reduce bone loss, relieve pain, and improve prognosis in PMOP patients. However, the action mechanism of EMFs in treatment of PMOP is still elusive. P2X7 receptors are ionotropic ATP-gated receptors. They are cation-selective channels with significant Ca2+ permeability. Also, osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and osteoblasts is enhanced by activation of P2X7 receptors. It is reported that EMFs affect osteoblast proliferation and differentiation through enhanced intracellular Ca2+ transients. Our previous study found that EMFs promoted expression of P2X7 receptors in BMSCs. Osteogenic differentiation of BMSCs induced by EMFs was decreased by blocking P2X7 receptors. Since high consistency is shown between the osteogenic differentiation and Ca2+ transients regulated by P2X7 receptors and EMFs, we hypothesize that P2X7 receptor is one of the key receptors in osteogenesis induced by EMFs. In the present study, we aim to determine whether P2X7 receptors take part in osteogenesis induced by EMFs in vitro and in vivo and identify the possible underlying mechanisms. It is expected that this study could broaden our understanding of biological effect of EMFs, and provide novel theory basis for the clinical application of EMFs in PMOP.

绝经后骨质疏松症（PMOP）及其相关骨折已成为一个严重的公共健康问题。临床试验显示电磁场可以减少PMOP病人的骨量丢失、缓解疼痛及改善预后，但其作用机制尚不明确。嘌呤受体P2X7是一类ATP门控离子通道受体，被激活时可引起Ca2+内流。此外，激活P2X7受体可促进成骨细胞和骨髓间充质干细胞（BMSCs）成骨分化。研究发现电磁场通过增加成骨细胞的Ca2+内流来促进其增殖和成骨分化。我们前期研究显示：电磁场可增加BMSCs的P2X7受体表达；封闭P2X7受体后，电磁场促进BMSCs成骨分化的能力下降。基于P2X7受体与电磁场的促成骨分化及介导的Ca2+内流作用高度吻合，我们推测P2X7受体可能是介导电磁场促成骨过程中的关键受体之一。本项目拟对P2X7受体在电磁场促成骨中的作用进行系统研究，并探索其分子机制。为进一步理解电磁场的生物学机制和在临床上应用电磁场治疗PMOP提供理论依据。

临床研究发现电磁场可以减少绝经后骨质疏松症（PMOP）病人的骨量丢失、缓解疼痛及改善预后，但其作用机制尚不明确。研究发现电磁场通过增加成骨细胞的Ca2+内流来促进其增殖和成骨分化。嘌呤受体P2X7是一类ATP门控离子通道受体，被激活时可引起Ca2+内流。我们推测P2X7受体可能是介导电磁场促成骨过程中的关键受体之一。本项目通过对低频正弦波电磁场促进成骨的最佳参数进行筛选，并研究其对骨代谢相关细胞的P2X7受体表达的影响。通过体内和体外实验分别验证P2X7受体在电磁场促成骨中的作用。检测电磁场通过P2X7受体激活的下游信号通路，并通过体内和体外实验分别验证筛选出的信号通路在电磁场促成骨中的作用。本研究发现：在成骨诱导条件下，1mT、7.5-75Hz的正弦波电磁场均可促进人骨髓间充质干细胞（h-BMSC）P2X7受体的表达，其中1mT、15Hz的电磁场作用最强。1mT、15Hz电磁场促进h-BMSC的RUNX2、ALP及OPN的表达，使磷酸化的Akt和GSK3β水平明显增加，并使胞内β-catenin表达增加。含有P2X7受体阻滞剂（A740003）的成骨诱导培养基诱导的BMSCs受电磁场刺激后，RUNX2、ALP及OPN的表达比对照组明显下降。单独使用A740003或PI3K/Akt 通路阻滞剂LY294002均使电磁场诱导的磷酸化Akt和GSK3β水平下降，同时使胞内β-catenin表达下降。在大鼠卵巢切除骨质疏松症模型（OVX）中，电磁场处理可以使OVX大鼠骨小梁体积增加30%，与P2X7受体激动剂可以使其增加50%。这些结果提示P2X7的激活可以增强电磁场对骨质疏松症的治疗作用。本研究结果为进一步理解电磁场的生物学机制和在临床上应用电磁场治疗骨质疏松提供理论依据。我们的研究结果还提示电磁场和P2X7激动剂联合治疗可作为治疗骨质疏松症的有效新靶点。