α-生育酚促进氧化应激状态下骨髓间充质干细胞的成骨分化

α-tocopherol promotes osteogenic differentiation of bone marrow mesenchymal stem cells under oxidative stress

目的:研究α-生育酚对氧化应激状态下骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的保护作用。方法:从4周龄大鼠体内提取原代BMSCs。CCK-8方法检测不同浓度的α-生育酚对过氧化氢(H_(2)O_(2))刺激的BMSCs细胞活力的影响。ROS荧光探针、流式分析和Western Blot评估α-生育酚对H_(2)O_(2)刺激的BMSCs细胞内活性氧水平的影响。Western Blot、RT-PCR、ALP和茜素红染色分析α-生育酚对H_(2)O_(2)刺激的BMSCs成骨分化的影响。结果:CCK-8结果表明α-生育酚维持了H_(2)O_(2)刺激的BMSCs的细胞活力,α-生育酚浓度为10μmol/L时,BMSCs活性维持的最好。ROS荧光探针、流式分析和Western Blot结果提示α-生育酚浓度为10μmol/L时显著降低了H_(2)O_(2)刺激的BMSCs细胞内ROS的表达。Western Blot和RT-PCR证明了α-生育酚促进了H_(2)O_(2)刺激的BMSCs成骨相关蛋白(ALP、RUNX2)和mRNA(Alp、Opn、Runx2)的表达。ALP和茜素红染色进一步验证了α-生育酚促进了氧化应激状态下BMSCs的成骨分化性能。结论:α-生育酚能够降低H_(2)O_(2)刺激的BMSCs细胞内氧化应激水平,维持氧化应激状态下的BMSCs细胞活性并促进其成骨分化能力。

Objective:To study the protective effect of α-tocopherol on bone marrow mesenchymal stem cells(BMSCs)under oxidative stress.Methods:Primary BMSCs were extracted from 4-week-old rats.CCK-8 method was used to detect the effect of different concentrations of α-tocopherol on cell viability of H_(2)O_(2)-stimulated BMSCs.ROS fluorescent probe,Flow cytometry assays and Western Blot evaluated the effect of α-tocopherol on reducing the intracellular reactive oxygen species levels in H_(2)O_(2)-stimulated BMSCs.Western Blot,RT-PCR,ALP and Alizarin red staining were used to analyze the effect of α-tocopherol on osteogenic differentiation of H_(2)O_(2)-stimulated BMSCs.Results:CCK-8 result showed that α-tocopherol maintained the cell viability of H_(2)O_(2)-stimulated BMSCs,the viability of BMSCs was best maintained at 10 μmol/L α-tocopherol.ROS fluorescence probe,Flow cytometry assays and Western Blot results suggested that 10 μmol/L α-tocopherol significantly reduced the intracellular ROS expression of H_(2)O_(2)-stimulated BMSCs.Western Blot and RT-PCR demonstrated that α-tocopherol promoted the expression of osteogenic associated proteins(ALP,RUNX2)and mRNA(Alp,Opn,Runx2)in H_(2)O_(2)-stimulated BMSCs.ALP and Alizarin red staining further confirmed that α-tocopherol promoted the osteogenic differentiation of BMSCs under oxidative stress.Conclusion:α-tocopherol can reduce the intracellular level of oxidative stress in H_(2)O_(2)-stimulated BMSCs,maintain the cellular viability of BMSCs under oxidative stress and promote its osteogenic differentiation ability.