维拉帕米在应力对体外骨髓间充质干细胞早期反应基因蛋白表达中作用的研究

Effects of verapamil on the immediate-early gene expression of bone marrow mesenchymal stem cells stimulated by mechanical strain

[目的]研究维拉帕米(Verapamil)在周期性牵张应力对体外骨髓间充质干细胞(MSCs)早期反应基因蛋白表达中的作用,从而进一步推断出Ca2+通道在细胞响应力学的信号传导途径中的作用。[方法]分离并培养骨髓间充质干细胞,利用体外细胞牵张应力装置对第3～6代细胞加载力学信号,在应力干预细胞前加及不加钙离子拮抗剂(维拉帕米),受力结束后,流式细胞术检测细胞内钙离子荧光强度,用RT-PCR以及免疫组化的方法检测各组细胞内c-fos、c-jun和c-myc基因蛋白表达的情况。[结果]加载力学信号后,细胞内钙离子浓度发生改变,c-fos、c-jun和c-myc基因蛋白的表达明显增强(P<0.01),经维拉帕米预处理后,以上生物学效应受到抑制(P<0.01)。[结论]在牵张应力作用下骨髓间充质干细胞Ca2+内流发生了变化,并且这种变化在骨髓间充质干细胞对周期性牵张应力的早期应答反应中起到了部分作用,可能依赖钙离子通道的活化来调控。

[ Objective]To study the effects of verapamil on the immediate-early genc（IEGs） expression of bone marrow mesenehymal stimulated by mechanical strain,and deduce the effects of calcium ion channel on the early responses of children bone marrow mesenchymal stem cells （MSCs）to mechanical strain in vitro. [ Method J MSCs were isolated and cultured. The 3 -6^th generational MSCs were stimulated in different time by mechanical and （or） verapamil（20μmol/1）. Flow cytometry was applied to measure intracellular free Ca^2＋ at once. Early-response genes/proteins （ c-los ,c-jun and c-myc） were examined by RT-PCR and immunocytochemical stain. [ Result ] The application of mechanical strain increased intracellular c-fos, c-jun and c-myc mRNA/protein levels. The resulting effect is partially inhibited before MSCs were preincubated with verapamil （P 〈0.01 ）. [ Conclusion] Mechanical strain could change intraeellular free calcium ion concentration of bone marrow mesenchymal stem cells,which was partially inhibited by verapamil,and the change playes a key role in early response of MSCs to mechanical loading.