摘要
该文探讨了低浓度过氧化氢(H_2O_2)对骨髓间充质干细胞增殖、迁移及其相关信号通路的影响,阐明了低浓度H_2O_2发挥生物学效应的分子机制,为临床应用提供了可靠的实验证据。通过差速贴壁分离培养小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)。流式细胞术鉴定第三代BMSCs表面标志物。采用随机数字表法分组,以不同低浓度H_2O_2(0、25、50、100、150、200μmol/L)处理BMSCs 24 h,应用甲氮甲唑蓝(MTT)法检测细胞增殖,流式细胞术检测干细胞表面标志物以及凋亡率,划痕实验和Transwell实验检测H_2O_2对细胞迁移能力的影响。Western blot检测细胞老化相关蛋白质p16和Cyclin D1、基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)与其受体CXCR4(CXC chomekine receptor 4)的表达以及相关下游信号通路PI3K/AKT/mTOR关键蛋白质的表达。流式细胞术检测结果显示,BMSCs细胞表面分化抗原CD29、CD44为阳性,CD34、CD45为阴性。MTT检测结果显示,与对照组相比,25和50μmol/L H_2O_2能有效促进BMSCs增殖(P<0.05)。划痕实验和Transwell实验检结果显示,50μmol/L H_2O_2处理细胞能促进其迁移能力。流式细胞术检测显示,25、50和100μmol/L H_2O_2对干细胞凋亡无影响,150和200μmol/L H_2O_2则显著促进细胞凋亡(P<0.01);25和50μmol/L低浓度H_2O_2处理干细胞能抑制老化相关蛋白质p16表达下降(P<0.05,P<0.01),相反,细胞周期蛋白Cyclin D1表达则显著升高(P<0.05,P<0.01),H_2O_2为200μmol/L时,p16表达上调(P<0.01),而Cyclin D1下调(P<0.01);同样,25~100μmol/L H_2O_2能增强细胞SDF-1和CXCR4的表达(P<0.05)以及上调下游信号通路关键蛋白质PI3K、AKT、mTOR的磷酸化水平(P<0.05)。结果表明,低浓H_2O_2度通过活化干细胞SDF-1/CXCR4信号轴,活化其下游PI3K/Akt/mTOR信号通路,促进干细胞增殖和迁移。
This work was aim to investigate the effect of low dose of H_2O_2 on the migration and proliferation of bone marrow mesenchymal stem cells（BMSCs）, and its mechanism in BMSCs. BMSCs isolated from mice by adherence of time interval difference were treated with different low dose of H_2O_2 for 24 h. The surface markers of BMSCs, such as CD34, CD45, CD29, CD44 and CXCR4, were identified by flow cytometry analysis. The experimental groups were divided by random digits method. The effect of H_2O_2 on the cell migration ability was detected by Transwell migration assay and scraping. The effect of H_2O_2 on cell proliferation was measured by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. The rate of BMSCs apoptosis was detected by flow cytometry analysis. The levels of SDF-1 and its receptor CXCR4, aging-related proteins and key proteins in the PI3 K/Akt/mTOR pathway were detected by Western blot. The results from flow cytometry analysis showed that BMSCs highly expressed CD29 and CD44, very lowly expressed CD34 and CD45 on their cell surfaces. Low dose of H_2O_2（25 and 50 μmol/L） effectively promoted BMSCs proliferation（P〈0.05）. The apoptosis rates of BMSCs in 25, 50 or 100 μmol/L H_2O_2 groups were no significant differences compared with the control. However, the rates of BMSCs apoptosis in 150 or 200 μmol/L H_2O_2 groups were significantly increased compared with the control（P〈0.01）. The results of Transwell migration assay and scraping showed that 50 μmol/L H_2O_2 significantly augmented the migration ability of BMSCs（P〈0.05）, which could be inhibited by CXCR4 antibody. In addition, we found that 25 or 50 μmol/L H_2O_2 downregulated the levels of aging-related protein p16（P〈0.05, P〈0.01）, inversely, upregulated the levels of pro-cell cycle protein Cyclin D1（P〈0.05, P〈0.01）. Simultaneously, 25-100 μmol/L H_2O_2 induced an obvious increase in the levels of SDF-1 and its receptor CXCR4, and its downstream key proteins phosphorylated, such
出处
《中国细胞生物学学报》
CAS
CSCD
2018年第1期47-56,共10页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81372059
81571912)
国家重点基础研究发展规划项目(批准号:2012CB518105)
重庆市基础与前沿研究计划院士专项(批准号:cstc2017zdcy-yszx0003)
重庆高校创新团队建设计划资助项目(批准号:CXTDX201601005)资助的课题~~
