长鳍金枪鱼多肽制备及其对H2O2诱导的张氏肝细胞的保护作用  

The Preparation of Thunnus alalunga Polypeptide and Its Protective Effect on the Injury of Chang Liver Cells Induced by H2O2

作　　者：艾杨洋 丁国芳 杨最素[1] 余方苗 唐云平 陈艳 黄芳芳 Ai Yangyang;Ding Guofang;Yang Zuisu;Yu Fangmiao;Tang Yunping;Chen Yan;Huang Fangfang(Food and Medicine School of Zhejiang Ocean University,Key Engineering Reseach Centers of Marine Organisms Medical Products,Zhoushan 316022,Zhejiang;Marine Fisheries Research Institute of Zhejiang Province,Zhoushan 316021,Zhejiang)

机构地区：[1]浙江海洋大学食品与医药学院/浙江省海洋生物医用制品工程技术研究中心,浙江舟山316022 [2]浙江省海洋水产研究所,浙江舟山316021

出　　处：《中国食品学报》2020年第3期81-89,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(81001393);国家海洋创新示范城市舟山项目(2016496);国家海洋计划项目(2015862);浙江省科技厅重大专项(2013C03036);浙江省自然科学基金项目(LS15H30001)。

摘　　要：目的:以长鳍金枪鱼废弃物为原料提取多肽,探究其对H2O2诱导的张氏肝细胞的保护作用。方法:以H2O2诱导张氏肝细胞损伤建立细胞模型组,以相对增殖率为指标筛选长鳍金枪鱼废弃物最适酶种,通过正交试验确定最佳酶解条件。酶解产物经超滤、阴离子交换色谱、凝胶过滤色谱和反相高效液相色谱方法纯化分离得到多肽,用噻唑蓝(MTT)比色法对其每步分离效果进行评价。多肽活性的评价是利用试剂盒方法测定张氏肝细胞上清液中AST、ALT、MDA、ADH和SOD的含量,倒置显微镜观察细胞形态及流式细胞仪检测多肽对H2O2诱导后的张氏肝细胞凋亡的影响。结果:最适酶种为胰蛋白酶,最佳酶解条件为料液比1∶3(g/mL),pH 9,加酶量900 U/g,温度45℃,时间5 h。经一系列纯化后制成多肽,命名为长鳍金枪鱼多肽。其氨基酸序列为Gly-Ala-Pro-Gly-Glu-Arg-Gly-Ser-Lys-Cys-Phe-Lys。经长鳍金枪鱼多肽作用于H2O2诱导的张氏肝细胞后,ALT、AST、ADH和MDA含量下降,SOD含量上升。通过流式细胞仪检测发现晚期凋亡细胞相比于模型组减少明显。结论:利用酶解方法提取的长鳍金枪鱼多肽对H2O2损伤的张氏肝细胞具有一定的保护作用。Objective:This study aimed to extract polypeptide from the castoff of Thunnus alalunga and explore its protective effects on the damaged Chang liver cells induced by H2O2.Methods:The castoff of T.alalungawas pretreated for enzymatic hydrolysis.The optimal enzyme was screened using relative growth rate as the indicator.The optimal enzymolysis condition was determined by orthogonal tests.After hydrolysis,the resulting product was orderly purified by the ultrafiltration,ion exchange chromatography,gel filtration chromatography and reversed-phase high-performance liquid chromatography,and MTT method was used to evaluate the purification capacity for each step.The activities of AST and ALT enzymes were assayed by kit according to the manufacturer’s instruction.Inverted microscope and the flow cytometric analysis observation were used to investigate the effects of polypeptide addition on the apoptosis of Chang liver cells induced by H2O2.Results:The optimal enzyme for enzymolysis was trypsin,and its corresponding optimal hydrolysis condition was determined as follow:solid-liquid ratio was 1∶3,pH value was 9,enzyme dose was 900 U/g,temperature was maintained at 45℃and held for 5 h.After a series of purification,pure polypeptide was harvested and its amino acid sequence was identified as Gly-Ala-Pro-Gly-Glu-Arg-Gly-Ser-Lys-Cys-Phe-Lys.With the addition of the identified polypeptide on Chang liver cells,the activities of ALT,AST,ADH and MDA were decreased but the activities of SOD were increased relative to control group without polypeptide during the period of H2O2 induction.The flow cytometry results demonstrated that the late apoptotic cell amount was significantly decreased relative to the control group.Conclusion:The identified T.alalunga polypeptide had a protective effect on the H2O2 induced Chang liver.

关 键 词：长鳍金枪鱼废弃物 多肽 H2O2损伤 张氏肝细胞 护肝 

分 类 号：TS254.9[轻工技术与工程—水产品加工及贮藏工程]