PEP-1-CAT抑制氧化应激条件下大鼠骨髓间充质干细胞的凋亡

PEP-1-CAT Inhibits Apoptosis of Mesenchymal Stem Cells under Oxidative Stress

目的:研究PEP-1-CAT融合蛋白转导入大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,MSC)的能力,以及其预处理对MSC在氧化应激损伤条件下凋亡的影响及其机制。方法:采用流式细胞仪检测MSC表面标记物CD29、CD90、CD45、CD34,并检测成脂肪细胞和成骨细胞的诱导分化能力。采用免疫荧光染色和Western blot检测融合蛋白His-CAT和His-PEP-1-CAT转导入MSC的能力。DAPI染色和流式细胞仪检测细胞的凋亡率,采用相应的试剂盒分别检测丙二醛(MDA)含量、乳酸脱氢酶(LDH)活性的改变和线粒体膜电位的变化。结果:MSC低表达CD34(1.78%)、CD45(1.41%),高表达CD29(94.3%)、CD90(95.5%),具有成脂、成骨多向分化的潜能。PEP-1-CAT转导入MSC中呈现时间和剂量依赖性特征,而His-CAT不能进入细胞内。与His-CAT组相比较,PEP-1-CAT融合蛋白预处理组MSC的凋亡率明显降低,LDH和MDA含量明显下降,减轻了因氧化应激引起的线粒体膜电位的下降。结论:PEP-1-CAT融合蛋白通过清除活性氧,维持线粒体膜电位的稳定,显著抑制了MSC在氧化应激损伤情况下的凋亡。

Objective To investigate the transduction ability of PEP-1-CAT into cultured rat bone marrow mesenchymal stem cells(MSC),as well as the effect on apoptosis and its mechanism of PEP-1-CAT pretreatment on oxidative stress injury in MSC.Methods MSC shape and surface markers(CD29,CD34,CD45 and CD90) assay were detected by inverted microscope and flow cytometric analysis,respectively.The multipotent differentiation of lipocyte and osteoblasts were identified by Oil red O and AgNO3 staining.CAT and PEP-1-CAT fusion protein with an N-terminal His-tag were purified by affinity chromatography,and their transduction ability were determined with immunofluorescence and Western blot.Apoptosis of MSC was evaluated with DAPI staining and quantitatively assayed with Annexin V and PI double staining by Flow cytometry.Maleic dialdehyde(MDA),lactate dehydrogenase(LDH) were simultaneously measured.The mitochondrial membrane potential of MSC was analyzed with JC-1 staining.Results Cultured MSC had the ability of multipotent differentiation,highly expressed CD29(94.3%) and CD90(95.5%),lowly expressed CD34(1.78%) and CD45(1.41%).PEP-1-CAT fusion protein was effectively transduced into MSC in a dose-and time-dependent manner.Compared to His-CAT group,the cells apoptosis rate,the contents of MDA and LDH were significantly decreased in PEP-1-CAT group.Consistent with its effect on MSC apoptosis,PEP-1-CAT inhibited the decrease of H2O2-attenuated mitochondrial membrane potential.Conclusion PEP-1-CAT could efficiently inhibit apoptosis of MSC under oxidative stress by scavenging reactive oxygen species and maintaining the stability of the mitochondrial membrane potential.