双齿围沙蚕多肽的制备及其抗肺癌A549细胞活性  

Anticancer Activity of a Novel Peptide Derived from Hydrolysates of Perinereies aibuhitensis against Lung Cancer A549 Cells

作　　者：贾盈露 丁国芳[1,2] 杨最素 余方苗 郑媛媛 吴宗泽 陈锐 JIA Yinglu;DING Guofang;YANG Zuisu;YU Fangmiao;ZHENG Yuanyuan;WU Zongze;CHEN Rui(Key Engineering Research Centers of Marineorganisms Medical Products, School of Food Science and Medical,Zhejiang Ocean University, Zhoushan 316022, China;Marine Fisheries Research Institute of Zhejiang, Zhoushan 316021, China)

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：《食品科学》2017年第12期27-35,共9页Food Science

基　　金：国家自然科学基金青年科学基金项目(81001393);2015年度国家星火计划项目(2015GA700044);国家海洋重大计划项目(2015862);浙江省科技厅重大专项(2013C03036);浙江省自然科学基金项目(LS15H30001);浙江省自然科学基金青年资金项目(LQ16H300001);舟山市科技计划项目(2015C31012)

摘　　要：探讨双齿围沙蚕酶解多肽制备的关键技术及其对肺癌A549细胞的作用。以增殖抑制率为指标,确定最佳酶种并根据单因素试验和正交试验确定其最佳酶解工艺。经超滤获得1~3 ku的酶解液,通过阴离子色谱、凝胶过滤色谱和制备色谱对其进行进一步的分离纯化。采用四甲基偶氮唑蓝法测定沙蚕多肽PAP对肺癌A549细胞的增殖抑制率并通过倒置显微镜、吖啶橙/溴化乙锭荧光染色及Hoechst荧光染色观察其细胞形态的变化。实验结果表明最佳酶种是碱性蛋白酶,其最佳酶解条件为:酶解温度50℃、pH 11、料液比1∶1(g/mL)、酶解时间6 h、加酶量300 U/g。经纯化获得的多肽命名为PAP,其氨基酸序列为Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe,且PAP对A549细胞的作用呈时间与剂量依赖关系,作用后细胞出现了凋亡的形态学特征。因此,PAP能明显抑制肺癌细胞A549增殖,可以诱导其发生凋亡而发挥抗肿瘤作用。The key steps for preparing antitumor peptide from enzymatic hydrolysates of Perinereies aibuhitensis wereinvestigated in the present work.The most suitable enzyme preparation was selected and the optimal hydrolysis conditionsallowing high inhibition of cancer cell proliferation were determined using one-factor-at-a-time method and orthogonalarray design.A fraction containing peptides with molecular weights of1?3ku was obtained by ultrafilation of hydrolysates,and it was further purified by sequential ion exchange chromatography,gel filtration chromatography and preparativechromatography.Then,lung cancer A549cells were used to test the anticaner effect of the purified peptide PAP bymethylthiazolyldiphenyl-tetrazolium bromide(MTT)assay.The morphological changes were observed through an invertedmicroscope,dual acridine orange/ethidium bromide(AO/EB)fluorescent staining and Hoechst fluorescent staining.Theresults showed that alcalase was found to be the optimal enzyme for the production of anticancer peptide and that the optimalhydrolysis conditions were determined as follows:temperature,50℃;pH value,11;solid-to-liquid ratio,1:1;hydrolysistime,6h;and enzyme dosage,300U/g.The peptide PAP was identified as Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe.Moreover,our results demonstrated that PAP suppressed the proliferation of A549cells in a time-and dose-dependentmanner with morphological features of apoptosis being observed.Therefore,our findings suggest that PAP could inhibit theproliferation of lung cancer A549cells and induced apoptosis.

关 键 词：双齿围沙蚕 多肽 抗肿瘤 A549细胞 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]