体外冲击波通过三磷酸腺苷诱导人骨髓间充质干细胞向成骨细胞分化

Extracorporeal shockwaves induce osteogenic differentiation of human mesenchymal stem cells by ATP release

目的研究体外冲击波是否通过三磷酸腺苷(ATP)激活P2X_7受体,诱导人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)向成骨细胞分化。方法培养hMSCs细胞,检测冲击波是否引起其向外释放ATP;通过检测碱性磷酸酶(ALP)活性、骨钙素表达和钙结节形成,判断骨化形成和钙质沉积;用实时定量PCR检测P2X_7受体的mRNA表达;用ATP水解酶、P2X_7受体的siRNA以及P2受体的抑制剂评估ATP释放和P2X_7受体在冲击波诱导hMSCs成骨分化中的作用。结果冲击波可引起细胞内ATP向外释放,冲击波和细胞外ATP能够诱导hMSCs向成骨分化,采用ATP水解酶、P2X_7受体的siRNA和抑制剂能够抑制冲击波引起的hMSCs成骨化作用。结论冲击波通过引起细胞内ATP向外释放,激活P2X_7受体传导信号通路,促进hMSCs向成骨细胞分化。本研究结果为冲击波促进骨折愈合和治疗骨不连疗法提供了理论依据。

Objective To investigate whether extracorporeal shockwave could induce differentiation of human mesenchymal stem cells （hMSCs） into osteoprogenitor cells by ATP release and the activation of P2X7 receptors. Methods Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase （ALP） activity, osteocalcin （OC） production, and calcium nodule formation. The mRNA expression of P2X7 receptors was determined with real-time RT-PCR. P2Xv-siRNA, apyrase, and P2 receptor antago- nists were used to evaluate the roles of ATP release and P2X7 receptors in shockwave-induced osteogenic hMSCs differentiation. Results Shockwave treatment released significant amounts of ATP from hMSCs. Shockwaves and exogenous ATP induced hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2XT-SiRNA or selective antagonists prevented osteogenic differentiation of hMSCs. Conclusions Shockwaves can contribute to osteogenic differentiation of hMSCs by realeasing cellular ATP that activate signaling. These research findings provide the theoretical basis for shockwave therapy in treating fracture healing and bone nonunion.