人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析

doi:10.7503/cjcu20150062

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (7): 1275.doi: 10.7503/cjcu20150062

人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析

潘丽英, 顾笑, 王承健, 强珊, 黄琳娟, 张英(), 王仲孚()

西北大学生命科学学院, 西部资源生物与现代生物技术教育部重点实验室, 西安 710069

收稿日期:2015-01-19 出版日期:2015-07-10 发布日期:2015-06-17
作者简介:联系人简介: 王仲孚, 男, 博士, 教授, 博士生导师, 主要从事糖生物学与糖工程学研究. E-mail:wangzhf@nwu.edu.cn;张英, 女, 博士, 讲师, 主要从事糖链的荧光标记和质谱分析研究. E-mail:zhangying@nwu.edu.cn
基金资助:
国家自然科学基金(批准号: 31370804, 31170773)和陕西省教育厅自然科学专项基金(批准号: 2013JK0714)资助

Comparative Analysis of O-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02^†

PAN Liying, GU Xiao, WANG Chengjian, QIANG Shan, HUANG Linjuan, ZHANG Ying^*(), WANG Zhongfu^*()

Key Laboratory for Western China Resource Biology and Biotechnology, Ministry of Education, College of Life Science, Northwest University, Xi’an 710069, China

Received:2015-01-19 Online:2015-07-10 Published:2015-06-17
Contact: ZHANG Ying,WANG Zhongfu E-mail:zhangying@nwu.edu.cn;wangzhf@nwu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.31370804, 31170773) and the Scientific Research Program Funded by the Department of Education of Shaanxi Province, China(No.2013JK0714)

摘要/Abstract

摘要：

以培养的原发性肝细胞癌HepG2细胞和正常肝细胞L02为研究对象, 用细胞裂解液提取总蛋白, 然后采用Carlson还原性β-消除法释放O-糖链, 以阳离子交换柱结合C₁₈柱纯化分离O-糖链, 用电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对O-糖链进行序列鉴定, 以β-环糊精为内标对2种细胞系的O-糖链进行定量比较分析. 结果表明, 在肝癌细胞系HepG2中检测到10种O-糖链, 正常细胞系L02中检测到9种O-糖链, 其中9种O-糖链是2种细胞系中共有的, 但HepG2中存在癌细胞中特有的缩短的O-糖链N1A1(NeuAc-GalNAc, sialyl Tn 抗原). t检验结果表明, HepG2与L02相比, 在检测到的10种O-糖链中有5种的含量具有极显著性差异(P<0.01), 2种的含量具有显著性差异(P<0.05).

关键词: 原发性肝癌细胞HepG2, 正常人肝细胞L02, O-糖链, 电喷雾电离质谱

Abstract:

HepG2(a primary hepatocellular carcinoma cell line) and L02(ones derived from normal liver tissue) cells were chosen as model cell lines for research. The O-glycans of the total proteins extracted from HepG2 and L02 cells were released by Carlson reductive β-elimination. The released O-glycans previously purified by Dowex 50 WX8-400 cation exchange resin and C₁₈ cartridges were identified by electrospray ionization mass spectrometry(ESI-MS)and MS/MS. For comparision studies, β-cyclodextrin was used as the internal standard for relative quantitative analysis of the O-glycans derived from HepG2 and L02 cells by MS. As results, 10 O-glycans were observed in HepG2 cell line and 9 O-glycans were detected in L02 cell line. Moreover, 9 O-glycans were observed in both HepG2 and L02 cells, wherears 1 truncated O-glycan assigned as H1A1(NeuAc-GalNAc, sialyl Tn antigen, ubiquitous in cancer cells), was only found in HepG2 cells. t-Test results show that 5 and 2 O-glycans in HepG2 cells have significant differences(P<0.01 and P<0.05, recpectively), when compared to those of L02 cells. Our studies show methodological significance in structural investigation of O-glycans expressed in hepatocellular carcinoma and early biomarker discovery in clinical diagnose.

Key words: HepG2 cell, L02 cell, O-glycans, ESI-MS

中图分类号:

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潘丽英, 顾笑, 王承健, 强珊, 黄琳娟, 张英, 王仲孚. 人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析. 高等学校化学学报, 2015, 36(7): 1275.

PAN Liying, GU Xiao, WANG Chengjian, QIANG Shan, HUANG Linjuan, ZHANG Ying, WANG Zhongfu. Comparative Analysis of O-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02^†. Chem. J. Chinese Universities, 2015, 36(7): 1275.

图/表 7

Fig.1 ESI-MS spectra of O-glycans derived from HepG2(A, C) and L02(B, D) cells under positive ion mode(A, B) and negative ion mode(C, D), respectively * Impurities.

Fig.2 MS/MS spectrum of O-glycans with m/z 1355.33(A) and the proposed structures(B)

Fig.3 MS/MS spectra(A, C) and proposed structures(B, D) of O-glycans with m/z 657.17(A,B) and m/z 699.25(C, D)

Fig.4 MS/MS spectrum(A) and proposed structure(B) of O-glycans with m/z 515.25

Fig.5 MS/MS spectra of O-glycans at m/z 1353(A) and its proposed structure(B)

Fig.6 Peak intensity ratio of O-glycans to β-cyclodextrin from HepG2 and L0210 O-glycans: a. H1N1; b. N1A1; c. H1N2; d. H1N1A1([M+H]+) and H1N1A1([M+Na]+); e. H2N2; f. H1N1A2; g. H2N2A1; h. H2N2A2; i. H1N1A1F1; j. H1N2A2F2. Significant difference: ** P<0.01; * P<0.05.

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人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析

Comparative Analysis of O-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02^†

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人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析

Comparative Analysis of O-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02†

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Comparative Analysis of O-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02^†