体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

Hypoxia and Serum Deprivation Induced Bone Mesenchymal Stem Cells apoptosis Via PI3K/Akt

目的:研究体外大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。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,骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。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对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(Phosphoinosi-tide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂(LY294002)组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制(P<0.05)。结论:PI3K/Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。

Objective： To investigate the protective effects of PI3K （Phosphoinositide-3kinase）/Akt （ProteinkinaseB,PKB） on the marrow-derived mesenchymal stem cells. Methods： Mononuclear cells were isolated from rat bone marrow by density-gradient centrifu- gation and were cultured on fibronectin-coated plates, supplied with bovine pituitary extract. BMSCs were identified by Morphology and Surface molecule markers of BMSCs. After the marrow-derived mesenchymal stem cells were under hypoxia and serum deprivation （hypoxia and SD）, the proportion of apoptosis and the protein expression of p-Akt in different groups were compared by Fluorescence-activated cell sorter （FACS） analysis and WESTERN BOLT. Results： ① The marrow-derived mesenchymal stem cells were successful cultured by detecting the morphology of the marrow-derived mesenchymal stem cells and the cell surface antigen by using flow eytometry assays. ② The proportion of apoptosis had significantly increased in the group under hypoxia and SD compared with that of group under non hypoxia and SD. The expression ofp-Akt increased significantly which indicated the activation of PI3K/Akt （P〈0.05）. The proportion of apoptosis had significantly decreased in the group under hypoxia and SD plus the inhibitor of PI3K/Akt compared with that in the group under hypoxia and SD alone. The protein expression of p-Akt significantly decressed, which indicated the inhibition of PI3K/Akt （P〈0.05）. Conclusion： PI3K/Akt had crucially protective effects on the marrow-derived mesenchymal stem cells under hypoxia and SD.