低浓度过氧化氢预处理增强骨髓间充质干细胞抗氧化应激损伤的能力

Preincubation with low-concentration hydrogen peroxide enhances anti-oxidative stress ability of bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞移植促进骨坏死区血管再生和骨重建,取得了一定疗效,但是骨坏死区氧化应激微环境极大地限制了移植后骨髓间充质干细胞的存活、增殖和成骨分化能力,降低了对骨坏死区的修复作用。目的:探讨低浓度过氧化氢预处理对骨髓间充质干细胞抗氧化应激损伤的影响。方法:取生长良好的第3代兔骨髓间充质干细胞,实验分为3组:空白对照组,高浓度过氧化氢持续处理组(500μmol/L过氧化氢处理24 h),低浓度过氧化氢预处理组(50μmol/L过氧化氢预处理8 h,恢复12 h,再用500μmol/L过氧化氢处理24 h)。根据实验分组条件进行过氧化氢处理,采用DCFH-DA荧光染料检测细胞活性氧水平,ELISA法检测各组细胞超氧化物歧化酶、过氧化氢酶活力,比色法检测各组细胞丙二醛水平,JC-1法检测细胞线粒体膜电位,CCK-8法检测细胞活力,流式细胞仪和TUNEL/DAPI双染法检测细胞凋亡。结果与结论:①与高浓度过氧化氢持续处理组相比,低浓度过氧化氢预处理组可有效减少骨髓间充质干细胞内活性氧产生(P <0.05);同时,低浓度过氧化氢预处理组超氧化物歧化酶、过氧化氢酶活力明显升高(P <0.05),丙二醛水平明显降低(P <0.05),线粒体膜电位损伤减轻(P <0.05),细胞存活率提高(P <0.05),细胞凋亡减少(P <0.05);②结果表明低浓度过氧化氢预处理可显著增强骨髓间充质干细胞抗氧化应激损伤能力。

BACKGROUND: Bone marrow mesenchymal stem cell transplantation promotes vascular regeneration and bone remodeling in osteonecrosis area, and has achieved certain effects. However, the oxidative stress microenvironment in osteonecrosis area greatly limits the survival, proliferation and osteogenesis of bone marrow mesenchymal stem cells after transplantation, reducing the repairing effect on the osteonecrosis area.OBJECTIVE: To investigate the effect of low-concentration hydrogen peroxide(H2O2) pretreatment on the anti-oxidative stress injury to bone marrow mesenchymal stem cells.METHODS: The third generation of well-growing bone marrow mesenchymal stem cells were divided into three groups: blank control group,high-concentration H2O2 treatment group(500 μmol/L H2O2 treatment for 24 hours), low-concentration H2O2 pretreatment group(50 μmol/L H2O2 pretreatment for 8 hours, recovery for 12 hours, and then 500 μmol/L H2O2 treatment for 24 hours). Cellular level of reactive oxygen species was detected by DCFH-DA fluorescent dye;superoxide dismutase and catalase activities were detected by ELISA;malondialdehyde content was detected by colorimetry;mitochondrial membrane potential was detected by JC-1;cell viability was detected by cell counting kit-8;and cell apoptosis was detected by flow cytometry and TUNEL/DAPI double staining.RESULTS AND CONCLUSION: Compared with the high-concentration H2O2 treatment group, the low-concentration H2O2 pretreatment group could effectively reduce the production of reactive oxygen species in the bone marrow mesenchymal stem cells(P < 0.05);meanwhile,the low-concentration H2O2 pretreatment group showed significantly increased superoxide dismutase and catalase activities(P < 0.05),decreased malondialdehyde content(P < 0.05), reduced mitochondrial membrane potential damage(P< 0.05), increased cell survival rate(P < 0.05) and decreased cell apoptosis(P < 0.05). These findings indicate that low-concentration H2O2 pretreatment could significantly enhance the ability of bone marrow mesenchymal stem cells to resist oxidative stress injury.