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【目的】探究过氧化氢(H2O2)对骨髓间充质干细胞(BMSCs)氧化应激损伤所致衰老的相关机制,摸索合适的H2O2衰老造模浓度,为后期的药物干预实验研究奠定基础。【方法】采用全骨髓贴壁法培养BMSCs,H2O2分别以100、200、300、400、500、600、700、800、1 000μmol/L浓度作用于BMSCs 2、12、24、48 h,采用四甲基偶氮唑盐(MTT)法检测细胞代谢活力;β-半乳糖苷酶染色判断细胞衰老的建立成功与否;采用反转录荧光定量技术(RT-q real-time PCR)观察衰老相关基因p16、p21、p53表达水平的变化。【结果】当H2O2浓度>700μmol/L时,BMSCs凋亡明显升高,而H2O2浓度<400μmol/L时,变化不明显,甚至有刺激BMSCs增殖的作用;以500μmol/L H2O2作用BMSCs 24 h后,β-半乳糖苷酶染色阳性率显著增加,p21、p16、p53基因表达显著升高,差异均具有统计学意义(P<0.05或P<0.01)。【结论】以500μmol/L H2O2浓度作用于BMSCs 24 h后,可造成BMSCs氧化损伤衰老模型,并可使衰老相关基因p16、p21、p53表达上调。
【Objective】 To explore the mechanism of hydrogen peroxide (H2O2) on the aging induced by oxidative stress in bone marrow mesenchymal stem cells (BMSCs) and to explore the appropriate concentration of H2O2 aging model, which lays the foundation for the experimental study of drug intervention in the later stage. 【Methods】 BMSCs were cultured by whole bone marrow adherent method. H2O2 was administered to BMSCs at the concentrations of 100, 200, 300, 400, 500, 600, 700, 800 and 1000 μmol / L for 2, 12, 24 and 48 h respectively. MTT assay was used to detect cell viability; β-galactosidase staining was used to determine the success of cell senescence; and the effects of aging were observed by RT-q real-time PCR Gene p16, p21, p53 expression changes. 【Results】 Apoptosis of BMSCs was significantly increased when H 2 O 2 concentration was> 700μmol / L, while H 2 O 2 concentration <400μmol / L did not change obviously, even stimulated the proliferation of BMSCs. After treated with 500μmol / L H2O2 for 24 h , β-galactosidase staining positive rate was significantly increased, p21, p16, p53 gene expression was significantly increased, the difference was statistically significant (P <0.05 or P <0.01). 【Conclusion】 Apoptosis of BMSCs can be induced by exposure to 500μmol / L H2O2 for 24 h, and the expression of p16, p21 and p53 may be up-regulated.