PEP-1肽介导人Cu,Zn-SOD转导入大鼠离体缺血再灌注损伤心脏

目的:构建原核表达载体pET15b-PEP-1-SOD1,观察PEP-1-SOD1融合蛋白在大鼠离体缺血再灌注损伤心肌组织内的转导能力和转导的目的蛋白是否具有酶活性。方法:构建原核表达载体pET15 b-SOD1和pET15b-PEP-1-SOD1,分别转化大肠杆菌,表达和纯化SOD1和PEP-1-SOD1融合蛋白。采用Langendorff灌流系统,大鼠离体心脏停灌30 min后复灌60 min建立缺血再灌注损伤模型。实验分为对照组,SOD1组,25,50,100μmol/L PEP-1-SOD1组。免疫荧光检测PEP-1-SOD1的转导效果,SOD试剂盒检测酶活性。结果:在荧光显微镜下,PEP-1-SOD1融合蛋白预处理组心肌组织中可见特异性的绿色荧光位于心肌细胞胞质内,25,50,100μmol/L组阳性率分别为32.6%,42.5%,60.9%。SOD1蛋白预处理组和对照组未见到绿色荧光。与对照组相比,PEP-1-SOD1各组SOD酶活性均显著增加(P<0.01),且SOD酶活性与PEP-1-SOD1浓度呈正相关,SOD1组与对照组之间无显著性差异(P>0.05)。结论:PEP-1-SOD1融合蛋白以天然有活性的形式穿透进入大鼠离体缺血再灌注损伤的心肌细胞,且具有剂量依赖性。转导入细胞内的PEP-1-SOD1蛋白具有酶活性。本研究为SOD1治疗由氧化应激所导致的各种疾病提供了一种有效的递送途径。

Transduction of Human Cu, Zn-Superoxide Dismutase Mediated by PEP-1 Peptide into Isolated Rat Hearts with Ischemia Reperfusion Injury

Objective: To construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and prepare the purified PEP-1-SOD1 fusion protein,and to investigate the transduction efficiency of the purified PEP-1-SOD1 fusion protein and the enzyme activity of the transduced PEP-1-SOD1 in the isolated rat hearts with ischemia reperfusion injury.Methods: The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 was transformed into BL21(DE3) to express and purify SOD1 and PEP-1-SOD1,respectively.With Langendorff perfusion system,the in vitro ischemia reperfusion injury model was established with the isolated rat hearts reperfused 60 min following 30 min cessation of perfusion.The isolated rat hearts were categorized into control,SOD1 and 25,50,100 μmol/L PEP-1-SOD1 groups.The transduction efficiency was evaluated with immunofluorescent microscopy and the enzyme activity of the transduced PEP-1-SOD1 was measured with the protocol of SOD detection kit.Results: The bright green fluorescence was observed within the cytoplasma of cardiomyocytes in PEP-1-SOD1 pretreatment groups,whereas no signal was observed in control and SOD1 groups. The percentage of positive cardiomyocytes in PEP-1-SOD1 groups at 25,50 and 100 μmol/L was 32.6%,42.5% and 60.9%,respectively.The enzyme activity of the transduced PEP-1-SOD1 was significantly increased in a dose-dependent manner compared with control group.However,there was no significant difference in the enzyme activity between SOD1 and control groups.Conclusion: PEP-SOD1 fusion protein with natural and active form could effectively be transduced into the isolated rat hearts with ischemia refperfusion injury in a dose-dependent manner.The transduced PEP-1-SOD1 has the SOD enzyme activity.These findings provide an efficient therapeutic delivery for SOD1 in the therapy of various diseases related oxidative stress.