细胞穿透肽PEP-1介导人铜，锌-超氧化物歧化酶转导入大鼠离体心脏及其对缺血再灌注损伤的保护作用

The role of PEP-1-SOD1 fusion protein on ischemia-reperfusion injury in isolated perfused rat hearts

目的采用离体心脏灌注模型，研究细胞穿透肽PEP-1介导入铜，锌-超氧化物歧化酶（Cu，Zn-SOD，SOD1）穿透心肌组织能力及其对心脏缺血再灌注损伤的保护作用。方法构建的原核表达载体pET15b-SOD1和pET15b-PEP-1-1SOD1，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30min后复灌60min建立缺血再灌注损伤模型。大鼠随机分为对照组，100μmol／L SOD1蛋白预处理组，25、50、100μmol／LPEP-1-SOD1蛋白预处理组。免疫荧光及Western blot检测PEP-1-SOD1的转导效果，超氧化物歧化酶（SOD）试剂盒检测转导的目的蛋白的SOD活性。测定心肌组织丙二醛（MDA）含量和复灌后15min内冠状动脉流出液肌酸激酶（CK）活性。脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测心肌细胞凋亡，复灌60min时红四氮唑（TTC）染色测定心肌梗死面积。结果免疫荧光及Western blot检测均显示PEP-1-SOD1以剂量依赖性方式转导入离体灌注的心肌组织内，SOD1蛋白预处理组心肌组织内未见到目的蛋白。对照组，SOD1组和25、50、100μmol／LPEP-1-SOD1处理组的SOD活性分别为（10．06±0．77），（10．59±0．71）和（32．29±1．42）、（43．16±1．16）、（55．14±1．59）U／mg蛋白，MDA含量分别为（1．48±0．19），（1．39±0．11）和（1．01±0．14）、（0．73±0．13）、（0．50±0．06）nmol／mg蛋白，CK活性分别为（1．73±0．58），（1．68±0．14）和（1．40±0．28）、（0．97±0．39）、（0．61±0．56）U／mg蛋白，心肌细胞凋亡率（AI）分别为（17．25±0．75）％，（16．63±1．07）％和（11．50±0．57）％、（6．50±0．63）％、（4．13±0．52）％，心肌梗死面积百分比分别为（55．13±2．18）％，（52．13±2．59）％和（33．88±2．06）％、（25．50±2．16）％、（15．38±1．14）％。与对照组和SOD1组比较，PEP-1-SOD13种剂量组的SOD活性、MDA含量、CK活性、心肌细胞凋亡率及心肌梗死面积百分比指标差异均有统计学意义（均P〈0．01）。说明PEP-1-SOD1蛋白预处理组SOD活性以剂量依赖性方式显著高于SOD1组，MDA含量、CK活性及心肌细胞凋亡率均明显低于SOD1组，心肌梗死面积小于SOD1组。结论PEP-1肽介导SOD1蛋白能直接以天然活性的形式高效穿透进入离体心肌组织，转导的PEP-1-SOD1融合蛋白对心肌缺血再灌注损伤具有明显的保护作用。

Objective The transduction efficiency of the purified PEP-1-SOD1 fusion protein and the effects of PEP-1-SOD1 fusion protein on ischemia reperfusion injury in the isolated perfused rat hearts were investigated. Methods The constructed pET15b-SOD1 and pET15b-PEP-1-SOD1 were transformed into BL21 （DE3） for expression and purification of SOD1 and PEP-1-SOD1, respectively. Isolated perfused rat hearts were subjected to 60 min of global ischemia and 30 min of reperfusion and treated with vehicle, 100 μmol/L SOD1 and 25, 50, 100μmol/L PEP-I-SOD1, respectively. The transduction efficiency was evaluated with immunofluorescent microscopy and Western blot. The enzyme activity of the transduced PEP- 1-SOD1 was measured with commercial SOD detection kit. The MDA content in myocardial tissue and the CK activity in coronary exudate at 15 rain after reperfusion were also measured. Cardiomyocyte apoptosis was detected with TUNEL. The infarct size was determined in isolated hearts 60 min after reperfusion with TIC staining. Results Immunofluorescent microscopy and Western blot demonstrated PEP-1-SOD1 was transduced into myocardial tissue in a dose-dependent manner, whereas SOD1 could not be detected in SOD1 group. SOD activity in control, SOD1 group, 25,50,100 I.Lmol/L PEP-I-SOD1 groups was （10. 06 ±0. 77） U/mgprot, （10.59 ±0.71） U/mg prot, （32. 29 ± 1.42） U/mg prot, （43.16 ± 1.16） U/mg prot, （55. 14 ±1.59）U/mg prot, respectively. MDA content in corresponding groups was （ 1.48 ± 0. 19） nmol/mg prot, （ 1.39 ± 0. 11 ） nmol/mg prot, （ 1.01 ± 0. 14 ） nmol/mg prot, （ 0. 73 ± 0. 13 ） nmol/mg prot, （ 0. 50 ± 0. 06） nmol/mg prot, respectively. CK activity in corresponding groups was （ 1.73 ± 0. 58） U/rag prot, （ 1.68 ± 0. 14） U/mg prot, （ 1.40 ± 0.28 ） U/rag prot, （ 0.97 ± 0. 39 ） U/mg prot, （0.61 ±0.56 ） U/rag prot, respectively. Cardiomyocyte apoptotic index in corresponding groups was （17. 25 ± 0. 75 ）%, （16. 63 ± 1.07）%, （11.50 ± 0. 57） U/mg prot, （6. 50 ± 0. 63） U/mg prot, （4. 13 ± 0. 52）%, repectively. The percentage of myocardial infarction area was （ 55.13 ± 2. 18 ） %, （ 52. 13 ± 2. 59 ） %, （ 33.88 ± 2. 06 ） %, （25.50 ±2. 16）%, （ 15.38 ± 1.14）%, respectively. Compared with control group and SOD1 group, all P 〈0.01 These results demonstrated the enzyme activity of the transduced PEP-I-SOD1 was significantly increased in a dose-dependent manner and the MDA content, CK activity, the eardiomyocyte apoptotic index and the infarct size was decreased siginificantly in PEP-I-SOD1 pretreatment groups compared with SODI group. Conclusion The native, biologically active form of PEP-SOD1 fusion protein could be effectively transduced into the isolated rat hearts subjecting ischemia reperfusion injury in a dose-dependent manner. The transduced PEP-I-SOD1 has protective effects on ischemia reperfusion injury in the isolated rat hearts.