摘要
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 transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell （HUVECs） and the effects on hydrogen-peroxide （H2O2 ）-induced oxidative stress injury in these cells. Methods With the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E. coli BL21 （ DE3 ） and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni^2＋ -NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 （0. 5 mmol/L） alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase （ LDH ） and malondialdehyde（MDA） contents were measured. Results The PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group （37.23% ± 5.68% vs. 100%, P〈0.05） , while LDH leakage （849.3 U/L±95.1 U/L） and MDA （8.23 nmol/L ±1.58 nmol/L） content were significantly higher than that in control group （540. 6 U/L ± 65.7 U/L and 2. 46 nmol/L ± 1.42 nmol/L, respectively, all P 〈 0. 05 ）. Preincubation with PEP-1-CAT proteins at various concentrations （0. 25 -2 μmol/L） significantly attenuated H2O2 -induced cell injury. Conclusion The PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protei
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
2006年第10期932-938,共7页
Chinese Journal of Cardiology
