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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。
Objective: To investigate the ability of PEP-1-SOD1 fusion protein to transfect into rat bone marrow mesenchymal stem cells (MSCs) and its protective effects on MSCs under hypoxia-free and serum-free conditions. Methods: Purified SOD1 and PEP-1-SOD1 protein were added into the third generation MSCs cultured and identified respectively. The changes of PEP-1-SOD1 penetrating into MSCs were analyzed by immunofluorescence and Western blot. The changes of xanthine oxidase Enzymatic analysis of SOD activity. PEP-1-SOD1 was pretreated with 2.5μmol / L PEP-1-SOD1 for 45 min, then treated with hypoxia and serum for 24 h. Annexin V / PI double staining was used to detect the apoptosis of MSCs Happening. Results: The PEP-1-SOD1 fusion protein could be transduced into MSCs in a dose- and time-dependent manner in the cytoplasm and nucleus. The apoptosis rate of MSCs was significantly decreased under hypoxia-free and serum-free conditions (P <0.05). CONCLUSION: PEP-1-SOD1 fusion protein can be efficiently and stably transduced into MSCs and has a significant protective effect on apoptosis of MSCs induced by hypoxia-free serum.