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目的:研究恒磁场对体外缺血缺氧培养条件下大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)凋亡的影响并探讨其作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,将骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过TUNEL检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中特定蛋白质的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②缺血/缺氧组与缺血/缺氧+磁场组比较,缺血缺氧组的凋亡率显著性增加,Akt磷酸化水平显著上升(P<0.05)。提示恒磁场可以使PI3K(Phosphoinositide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活而抑制凋亡的发生。结论:恒磁场通过激活PI3K/Akt信号通路抑制体外缺血缺氧条件下培养的骨髓间充质干细胞的凋亡。
Objective: To investigate the effect of constant magnetic field on the apoptosis of Bone marrow-derived mesenchymal stem cells (BMSCs) under hypoxia-ischemia in vitro and its mechanism. Methods: Rat bone marrow was harvested and mononuclear cells (MNCs) were isolated by density gradient centrifugation. BMSCs were cultured and subcultured in vitro by PEX-induced amplification of bone marrow-derived mesenchymal stem cells (MSCs). After MSCs were identified by surface morphology and flow cytometry, bone marrow mesenchymal stem cells (BMSCs) were cultured under hypoxia and hypoxia conditions. TUNEL assay was used to compare the apoptosis rates of different groups of cells and Western blotting (western blot) to observe changes in a particular protein in the cell. Results: ①The surface markers of MSCs were detected by morphological observation and flow cytometry, which indicated that MSCs were cultured successfully. ② Compared with ischemic / hypoxic group and ischemic / hypoxic group, the apoptosis rate of ischemia / hypoxia group increased significantly, and Akt phosphorylation increased significantly (P <0.05). It is suggested that constant magnetic field can activate the PI3K (Phosphoinositide-3kinase) / Akt (ProteinkinaseB, PKB) signaling pathway to inhibit apoptosis. CONCLUSION: The constant magnetic field inhibits the apoptosis of BMSCs cultured in vitro by activating the PI3K / Akt signaling pathway.