hPP10-Cu,Zn-SOD融合蛋白的制备、穿膜效应及其抗氧化、抗炎症功效  

Cell membrane-penetrating capacity of hPP10-Cu,Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity

作　　者：张洁 姚君妍 杨英桂 王飞[6] 郑清友 李欣 柳长柏[2] ZHANG Jie;YAO Junyan;YANG Yinggui;WANG Fei;ZHENG Qingyou;LI Xin;LIU Changbai(Yuncheng Vocational Nursing College,Yuncheng 04400,China;China Three Gorges University,Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,Yichang 443002,China;Anesthesia Surgery Center,Shenzhen Hospital of Southern Medical University,Shenzhen 518100,China;Department of Urology,Shenzhen Hospital of Southern Medical University,Shenzhen 518100,China;Clinical Innovation and Research Center,Shenzhen Hospital of Southern Medical University,Shenzhen 518100,China;Department of Urology,Hainan Provincial People's Hospital,Haikou 570100,China)

机构地区：[1]运城护理职业学院,山西运城04400 [2]三峡大学肿瘤微环境与免疫治疗湖北省重点实验室,湖北宜昌443002 [3]南方医科大学深圳医院麻醉手术中心,广东深圳518100 [4]南方医科大学深圳医院泌尿外科,广东深圳518100 [5]南方医科大学深圳医院临床医学创新中心,广东深圳518100 [6]海南省人民医院泌尿外科,海南海口570100

出　　处：《南方医科大学学报》2024年第6期1059-1069,共11页Journal of Southern Medical University

基　　金：国家自然科学基金(81902788);深圳市科技计划(JCYJ20210324130801004);海南省重点研发项目基金(社发方向,SQ2021SHFZ0739)。

摘　　要：目的 探究人源性细胞膜穿透肽hPP10携带人源性抗氧化蛋白Cu,Zn-SOD的穿膜效应并观察其抗氧化、抗炎功效。方法利用RT-PCR技术扩增人源性SOD基因片段、酶切连接法构建重组质粒pET15b-Cu,Zn-SOD,pET15b-hPP10-Cu,Zn-SOD;利用镍柱亲和层析法、分子筛方法纯化Cu,Zn-SOD、hPP10-Cu,Zn-SOD融合蛋白并利用Western blotting法鉴定其正确性;利用免疫荧光法、荧光共定位实验、Western blotting法鉴定hPP10-Cu,Zn-SOD在细胞中的穿膜效应;利用Western blotting法鉴定hPP10-Cu,Zn-SOD融合蛋白穿膜时间梯度和浓度梯度效应;利用SOD酶活性测定试剂盒检测hPP10-Cu,Zn-SOD穿膜后的SOD酶活性;利用MTT法检测hPP10对细胞活性的影响。利用H2O2建立HEK293氧化应激细胞模型,通过流式细胞分析术(FCM)分析hPP10-Cu,Zn-SOD穿膜后细胞凋亡情况;并RT-qPCR分析hPP10-Cu,Zn-SOD穿膜后凋亡相关因子的表达情况;通过ROS检测分析hPP10-Cu,Zn-SOD穿膜后抗氧化能力。TPA诱导建立小鼠耳部炎症模型(5只/组,共4组),然后RT-qPCR分析hPP10-Cu,Zn-SOD穿膜后,NFκB,IL-1β、IL-6、TNFα因子的转录情况;Western blotting检测NFκB p65、p-NFκB p65、IL-1β、TNFα基因的表达;免疫组化分析炎性因子p-NFκB p65、TNFα基因的表达。结果 成功构建了重组质粒pET15b-Cu,Zn-SOD,pET15b-hPP10-Cu,Zn-SOD,并获得Cu,Zn-SOD蛋白和hPP10-Cu,Zn-SOD融合蛋白;免疫荧光试验结果表明5μmol/L浓度的hPP10-Cu,Zn-SOD转导HEK293细胞具有明显的穿膜效应;荧光共定位实验显示融合蛋白hPP10-Cu,Zn-SOD入胞后定位在细胞膜内,部分蛋白定位在细胞核内;Western blotting实验结果表明hPP10-Cu,Zn-SOD转导Hela(P<0.05),HEK293(P<0.01)细胞具有浓度依赖性,细胞内hPP10-Cu,Zn-SOD存在可持续24 h;5μmol/L的hPP10-Cu,Zn-SOD转导细胞具有较好的抗氧化活性(P<0.01);MTT结果显示高达10μmol/L浓度的hPP10-Cu,Zn-SOD对于细胞活力的影响小。在氧化应激模型中,FCM结果显示hPP10-Cu,Zn-SOD融合蛋�Objective To investigate the cell membrane-penetrating capacity of human cell-penetrating peptide hPP10 carrying human antioxidant protein Cu-Zn superoxide dismutase(Cu,Zn-SOD)and assess the antioxidant and anti-inflammatory activity of these fusion proteins.Methods The fusion protein hPP10-Cu,Zn-SOD was obtained by genetic engineering and identified by Western blotting.The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay,fluorescence colocalization assay and Western blotting,its SOD enzyme activity was detected using a commercial kit,and its effect on cell viability was assessed with MTT assay.In a HEK293 cell model of H2O2-induced oxidative stress,the effect of hPP10-Cu,Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR,and its antioxidant effect was assessed using reactive oxygen species(ROS)assay;its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR,Western blotting and immunohistochemistry.Results The fusion protein hPP10-Cu,Zn-SOD was successfully obtained.Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells,localized both in the cell membrane and the cell nuclei after cell entry.hPP10-Cu,Zn-SOD at the concentration of 5μmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10μmol/L.The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation.Conclusion The fusion protein hPP10-Cu,Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity,demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu,Zn-SOD in the development of skincare product

关 键 词：人源性细胞膜穿透肽hPP10 铜锌超氧化物歧化酶Cu Zn-SOD 抗氧化 抗炎 酶活力测定 

分 类 号：R914[医药卫生—药物化学]