PEP-1-SOD1融合蛋白预处理对大鼠骨髓间充质干细胞在缺氧无血清条件下的保护作用

目的:研究PEP-1-SOD1融合蛋白转导入大鼠骨髓间充质干细胞(MSCs)的能力及其在缺氧无血清条件下对MSCs的保护作用。方法:将纯化后的SOD1和PEP-1-SOD1蛋白分别加入到培养及鉴定后的第3代MSCs中,免疫荧光法和Western blot分析PEP-1-SOD1穿透入MSCs的变化,黄嘌呤氧化酶法分析其SOD酶活性。将2.5μmol/L PEP-1-SOD1预处理MSCs 45 min后去除PEP-1-SOD1,缺氧无血清条件下再处理24 h,Annexin V/PI双染标记,流式细胞术分析MSCs凋亡情况。结果:PEP-1-SOD1融合蛋白可以转导入MSCs,呈时间和剂量依赖性分布于胞浆和胞核,缺氧无血清条件下MSCs凋亡率明显降低(P<0.05)。结论:PEP-1-SOD1融合蛋白可以高效、稳定转导入MSCs,并对缺氧无血清所诱导的MSCs凋亡具有显著保护作用。

The Protective Effect of PEP-1-SOD1 Preconditioning Under Hypoxia and Serum Deprivation Condition in Rat Bone Marrow Mesenchymal Stem Cells

Objective To investigate the ability of PEP-1-SOD1 fusion protein transducting into rat bone marrow mesenchymal stem cells(MSCs),and its protective effects on MSCs under hypoxia and serum deprivation condition.Methods MSCs were cultured and characterized,the purified PEP-SOD1 and SOD1 was added in the third generation MSCs.The localization of PEP-1-SOD1 in MSCs was detected by immunofluorescecnce method,the PEP-1-SOD1 levels in MSCs were detected by semi-quantity Western blot.The activity of PEP-1-SOD1 in MSCs was detected by xanthine oxidase method.MSCs were pre-treated with 2.5μmol/L PEP-1-SOD1 for 45 minutes,and the cells were cultured under hypoxia and serum deprivation condition.The apoptosis of cells pretreated or untreated with PEP-1-SOD1 were analyzed by flow cytometry with annexin V FITC/PI double staining after 24 hours.Results PEP-1-SOD1 fusion protein could be transduced into MSCs in time and dose dependentand manner,distributed in cytoplasm and nuclei.The PEP-1-SOD1 preconditioning could significantly decreased the apoptosis inducing by hypoxia and serum deprivation(P<0.05).Conclusion PEP-1-SOD1 fusion protein could be efficiently transduced into MSCs,and protect the MSCs apoptosis from hypoxia and serum deprivation condition.