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目的探讨钙调神经磷酸酶(CaN)-T细胞核因子(NFAT)信号通路在应力介导的成肌细胞凋亡中的作用。方法构建成肌细胞体外培养—力学刺激模型,利用多通道应力加载系统对细胞加载不同时间的周期性张应力,加入CaN的特异性抑制剂环孢素(CsA)作为对比。采用Hoechst 33258染色法和流式细胞术检测成肌细胞凋亡情况,实时聚合酶链式反应检测CaN和NFAT mRNA的表达情况,蛋白质印迹法检测NFAT3的蛋白含量。结果随加力时间的延长,细胞凋亡逐渐增加,CaN亚基CnA、CnB及NFAT3的mRNA表达及NFAT3蛋白含量逐渐升高;加入CsA后,细胞凋亡减少,CnA、NFAT3的mRNA表达及NFAT3的蛋白含量明显减少。结论周期性张应力可以诱导成肌细胞发生凋亡;CaN-NFAT信号通路可能参与了周期性张应力诱导的成肌细胞凋亡。
Objective To investigate the role of calcineurin (CaN) -T nuclear factor (NFAT) signaling in stress-mediated myoblast apoptosis. Methods The in vitro culture-mechanical stimulation model of myoblasts was constructed. The cyclic tensile stress of cells was loaded by multi-channel stress loading system at different times, and cyclosporine (CsA), a specific inhibitor of CaN, was added as a contrast. The apoptosis of myoblasts was detected by Hoechst 33258 staining and flow cytometry. The expression of CaN and NFAT mRNA was detected by real-time polymerase chain reaction. The protein content of NFAT3 was detected by Western blotting. Results The apoptosis of CnA, CnB and NFAT3 increased and the content of CaN subunit increased gradually with the time prolonging. The apoptosis of CnA, NFAT3 mRNA and the expression of NFAT3 The protein content decreased significantly. Conclusions Cyclic tensile stress can induce myoblast apoptosis. CaN-NFAT signaling pathway may be involved in the cyclic tension-induced myoblast apoptosis.