PEP-1介导血红素加氧酶-1导入肝细胞的实验研究  

Transduction of heme oxygenase-1 mediated by PEP-1 peptide into L02 hepatocytes

作　　者：曾天才[1] 王卫星[1] 陈先祥[2] 唐俊明[2] 郭凌郧[2] 郑飞[2] 张林菲[2] 

机构地区：[1]武汉大学人民医院,湖北武汉430060 [2]湖北医药学院附属人民医院,湖北十堰442000

出　　处：《西部医学》2011年第3期411-414,418,共5页Medical Journal of West China

基　　金：湖北省教育厅科学技术研究计划重点项目(编号:D20082405)

摘　　要：目的采用体外培养的L02肝细胞株,研究PEP-1肽介导血红素加氧酶-1(PEP-1-HO-1)穿透肝细胞的能力。方法实验分为PEP-1-HO-1组和HO-1组,用基因工程的方法制备并纯化PEP-1-HO-1融合蛋白,将纯化的目的蛋白及HO-1蛋白加入培养的L02肝细胞,通过免疫荧光及western blot来分析其转导能力。结果经测序分析证实,原核表达质粒pET15b-PEP-1-HO-1重组成功。SDS-PAGE及western blot结果表明,PEP-1-HO-1在Rosetta(DE3)pLysS菌中获得了高效表达,经Ni2+-NTA-树脂柱亲和层析纯化得到了融合蛋白PEP-1-HO-1,将其加入到体外培养的L02肝细胞培养基中,发现它能以剂量依赖及时间依赖的方式转导入细胞内,并能持续稳定表达48h,免疫组化显示其主要分布于胞质和胞核中。结论 PEP-1-HO-1融合蛋白能高效地转导入培养肝细胞,为进一步研究肝细胞氧化应激损伤防治的动物模型实验提供了新的途径。Objective To investigate the transduction ability of PEP-1-HO-1 fusion protein whit L02 hepatocytes.Methods The study was divided into HO-1 treated group and PEP-1-HO-1 treated group.The constructed pET15b-PET-HO-1 was transformed into Rosetta（DE3） pLysS host bacteria which was induced by IPTG to obtain the fusion protein PEP-1-HO-1.The expressed PEP-1-HO-1 were purified with Ni2＋-resin affinity chromatography.Then the purified protein and HO-1 were added into cultured L02 hepatocytes.The transduction ability of PEP-1-HO-1 fusion protein into cells was analyzed by Western blotting and using immunofluorescence to evaluate the distribution of transduced fusion protein in L02 hepatocytes.Results The pET15b-PET-HO-1 was ligated successfully.SDS-PAGE and Western blotting demonstrated the fusion protein could successfully expressed by Rosetta（DE3）pLysS host bacteria and it was purified with Ni2＋-resin affinity chromatography.The result of immunofluorescence showed that t he PEP-1-HO-1 fusion protein could be transduced into L02 hepatocytes with native activity.The study showed that PEP-1-HO-1 fusion protein could enter L02 hepatocytes in a dose and time-dependent mannner when added into culture medium exogenously.And the fusion protein was stable for 48 hours once inside the cells.Conclusion The PEP-1-HO-1 fusion protein can efficiently penetrate the cultured L02 hepatocytes,which provides a new strategy for prevention and treatment of Hepatic ischemia-reperfusion injury whit HO-1.

关 键 词：血红素加氧酶 PEP-1肽 蛋白转导 肝细胞 

分 类 号：R-33[医药卫生]