摘要
目的:探讨低氧预处理对老年人骨髓间充质干细胞(h BM-MSCs)的保护作用,为提高老年自体干细胞移植治疗效果提供实验支持。方法:老年h BM-MSCs于低氧培养箱中培养24 h进行低氧预处理,实验分为年轻h BM-MSCs组(young组),老年h BM-MSCs组(old组)及低氧预处理老年h BM-MSCs组(old+hypoxia组)。300μmol/L H_2O_2作用30 min建立细胞氧化应激模型,50μmol/L LY294002作用2 h阻断PI3K/AKT信号通路,Brd U掺入实验检测细胞增殖能力;CCK-8法检测细胞活力,Western blot检测凋亡相关蛋白Bax、Bcl-2表达水平和AKT磷酸化水平。结果:Brd U掺入实验显示低氧预处理的老年h BM-MSCs细胞阳性率为39.85%±3.45%,与old组相比增殖能力显著提高(P<0.05)。300μmol/L H_2O_2作用30 min诱导细胞氧化应激后,old+hypoxia组与old组比较,细胞活力显著提高(P<0.05),凋亡相关蛋白Bax表达量显著降低(P<0.05),抑制凋亡的Bcl-2蛋白表达量显著增高(P<0.05),且AKT磷酸化水平显著增高,差异有统计学显著性(P<0.05);应用LY294002抑制PI3K/AKT信号通路后,细胞活力下降(P<0.05)。结论:低氧预处理可以通过激活AKT信号通路提高老年人骨髓间充质干细胞活力及增殖能力。
AIM： To investigate the protective effect of hypoxic preconditioning on human bone marrow mesenchymal stem cells（ h BM-MSCs）,and to provide basic experimental support for more effective autologous stem cell transplantation in aged patients. METHODS： The old h BM-MSCs were subjected to hypoxic preconditioning using a hypoxia incubator chamber for 24 h. The cells were divided into young group,old group and old ＋ hypoxia group（ with 24 h hypoxic preconditioning）. Hydrogen peroxide（ H_2O_2,300 μmol/L） was applied to simulate the oxidative stress. The cells were treated with 50 μmol / L LY294002 for 2 h to inhibit PI3 K / AKT pathway. Brd U incorporation and CCK-8 assay were used for analyzing the cell proliferation and viability. The protein levels of Bax,Bcl-2 and p-AKT were measured by Western blot. RESULTS： Brd U-positive cells,which represented the cell proliferation,and the cell viability were significantly increased in old ＋ hypoxia group compared with old group（ P〈0. 05）. The protein level of Bax decreased（ P〈0. 05） and Bcl-2 increased（ P〈0. 05） in old ＋ hypoxia group compared with old group after using 300 μmol / L H_2O_2 simulate. the oxidative stress. The phosphorylation of AKT was enhanced by hypoxic preconditioning in old group（ P〈0. 05）. The protective effect of hypoxic preconditioning on the cell survival was decreased after treated with LY294002（ inhibitor of the PI3 K / AKT pathway）（ P〈0. 05）. CONCLUSION： Hypoxic preconditioning increases the survival and proliferation of old hB M-MSCs by activation of AKT pathway.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第5期912-916,共5页
Chinese Journal of Pathophysiology
基金
山西医科大学青年基金资助项目(No.02201002)
山西医科大学基础医学院331基金资助项目(No.201217)
