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目的采用体外培养的人脐静脉内皮细胞株(CRL-1730),研究 PEP-1肽介导人过氧化氢酶(CAT)穿透细胞的能力,并探讨 PEP-1-CAT 对过氧化氢(H_2O_2)诱导的内皮细胞氧化应激损伤是否有保护作用。方法构建原核表达质粒 pET15b-PEP-1-CAT,将其在基因工程菌中表达出 N 端带有6个组氨酸“标签”(His-tag)的重组蛋白,并通过金属镍螯合亲和层析对其进行纯化,将纯化得到的PEP-1-CAT 融合蛋白加入体外培养的人脐静脉内皮细胞,通过 Western blot 来分析其转导能力,同时对转导人细胞内的蛋白进行酶活性检测。然后以0.5 mmol/L H_2O_2处理细胞建立内皮细胞氧化应激损伤的模型,检测不同浓度的 PEP-1-CAT 蛋白对氧化应激下细胞存活率、乳酸脱氢酶(LDH)活性及丙二醛(MDA)含量的影响。结果 PEP-1-CAT 融合蛋白能以时间、剂量依赖的方式高效转导进入细胞内。正常内皮细胞 LDH 活性和细胞存活率分别为(540.6±65.7)U/L和100%;H_2O_2处理组 LDH活性显著高[(849.3±95.1)U/L,P<0.01],细胞存活率明显低[(37.23±5.68)%,P<0.01],MDA含量亦较正常组显著高[(8.23±1.58)nmol/L 比(2.46±1.42)nmol/L,P<0.01]。用不同浓度的PEP-1-CAT 对细胞进行预保护,均可显著提高细胞存活率、降低 LDH 释放量,并提高细胞的抗氧化能力,表现为 MDA 含量较 H_2O_2处理组低(P<0.05或<0.01)。结论 PEP-1-CAT 融合蛋白能以天然活性形式高效转导入人脐静脉内皮细胞,且转导的蛋白能有效对抗氧化应激损伤。这种蛋白转导方式,为用 CAT 防治各种与氧化应激损伤有关的疾病提供了一个新的治疗途径。
Objective To investigate the ability of PEP-1 peptide to mediate the penetration of human catalase (CAT) into human umbilical vein endothelial cells (CRL-1730) in vitro and to investigate the effect of PEP-1-CAT on hydrogen peroxide H 2 O 2) induced endothelial cell oxidative stress injury whether there is a protective effect. Methods The prokaryotic expression plasmid pET15b-PEP-1-CAT was constructed and expressed in E.coli. The recombinant protein was expressed on the N-terminus with 6 histidine tagged His-tag, And purified by chromatography. The purified PEP-1-CAT fusion protein was added into human umbilical vein endothelial cells cultured in vitro, and the transduction ability was analyzed by Western blot. At the same time, the protein in transduced human cells was subjected to enzyme Activity test. Then the oxidative stress injury model of endothelial cells was established by treating cells with 0.5 mmol / L H 2 O 2, and the effects of different concentrations of PEP-1-CAT on cell viability, lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content of the impact. Results The PEP-1-CAT fusion protein efficiently transduced into cells in a time-and dose-dependent manner. The LDH activity and cell viability of normal endothelial cells were (540.6 ± 65.7) U / L and 100%, respectively. H 2 O 2 treatment group had significantly higher LDH activity (849.3 ± 95.1) U / L, P <0.01] [(37.23 ± 5.68)%, P <0.01]. MDA content was also significantly higher than that of the normal group [(8.23 ± 1.58) nmol / L vs (2.46 ± 1.42) nmol / L, P <0.01; Pretreatment of cells with different concentrations of PEP-1-CAT significantly increased cell viability, decreased LDH release, and increased cellular antioxidant capacity as compared with H 2 O 2 -treated mice (P <0.05 or P <0.05) <0.01). CONCLUSION: The PEP-1-CAT fusion protein can be efficiently transduced into human umbilical vein endothelial cells in its natural active form and the transduced protein is effective against oxidative stress injury. This method of protein transduction provides a new therapeutic approach for the prevention and treatment of various diseases associated with oxidative stress injury with CAT.