人肝癌细胞系HepG2与HepG2.2.15差异HLA结合肽的分离与鉴定  

Identification of differential HLA-binding peptide between two hepatoma cell lines HepG2 and HepG2.2.15

作　　者：陈黎[1] 谢兴旺[1] 张恒辉[1] 费然[1] 丛旭[1] 魏来[1] 陈红松[1] 

机构地区：[1]北京大学人民医院肝病研究所,北京市100044

出　　处：《世界华人消化杂志》2008年第21期2343-2348,共6页World Chinese Journal of Digestology

基　　金：新世纪优秀人才支持计划资助项目;No.985-2-099-113;国家重点基础研究(973)计划资助项目;No.2007CB512906~~

摘　　要：目的:分离并鉴定整合表达HBV的人肝癌细胞系HepG2.2.15及其母细胞系HepG2的差异HLAⅠ类分子结合肽,寻找与HBV感染相关的HLA结合肽.方法:洗涤法得到HepG2和HepG2.2.15细胞表面的HLAⅠ类分子及与其结合的肽段.效液相色谱(HPLC)对两种细胞的洗脱肽段进行分离和图谱比较,挑选出HepG2.2.15特有的峰段进行纳升电喷雾串联质谱(nano ESI-MS/MS)分析,结合MASCOT数据库检索和从头测序,分析多肽的序列和来源.后采用RT-PCR法进行mRNA表达检测.结果:HPLC对比显示HepG2和HepG2.2.15的HLA结合肽存在显著差异.利用nanoESI-MS/MS技术从HPLC差异峰段中分离鉴定出一条来源于已知蛋白人烯醇酶-1(enolase1,ENO1)的多肽SPDDPSRYISPDQ.RT-PCR结果显示,ENO1在HepG2和HepG2.2.15细胞中均有表达,且在HepG2.2.15细胞中表达明显高于HepG2细胞.结论:来自ENO1的多肽SPDDPSRYISPDQ能够被HLAⅠ类分子提呈到HepG2.2.15细胞表面,且ENO1 mRNA在HepG2.2.15细胞中的表达显著高于HepG2细胞.提示HBV感染可能引起ENO1表达上调.AIM： To screen the differential HLA-binding peptides between HepG2 and HepG2.2.15 cell lines, and to find some HLA-binding peptides correlated with hepatitis B virus （HBV） infection. METHODS： HepG2 and HepG2.2.15 cells were harvested （10s cells）, and the peptides were isolated from the cell membrane by mild acid elution, respectively. Then the mixture of peptides was fractionated by high performance liquid chromatography （HPLC） and the differential fractions only expressed in HepG2.2.15 cell line were identified by nanoESI-MS/MS analysis.Bioinformatic analysis and MASCOT index were used to investigate the sequence and source of the peptides. Finally the expression of mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： HPLC fractions were markedly different between HepG2 and HepG2.2.15 cells. A peptide, SPDDPSRYISPDQ, from enolase 1 （ENO1） was obtained, which was only expressed in HepG2.2.15 cells, by nanoESI-MS/MS analysis. The result of RT-PCR confirmed that ENO1 expression was significantly higher in HepG2.2.15 cells than that in HepG2 cells. CONCLUSION： A human peptide SPDDPSRYISPDQ from ENO1 can be presented on the surface of HepG2.2.15 cells by HLA, and ENO1 mRNA expression is significantly higher in HepG2.2.15 than that in HepG2, suggesting that HBV infection may cause the up-regualtion of ENO1 expression.

关 键 词：HEPG2细胞 HEPG2.2.15细胞 乙型肝炎病毒 烯醇酶-1 高效液相色谱 质谱 

分 类 号：R735.7[医药卫生—肿瘤]