乳腺癌细胞系N-糖基化特征性糖链的分析
摘要
细胞质膜是细胞与外界进行物质与信息交换的场所,近年来发现,细胞表面糖蛋白是许多细胞质膜功能重要的执行者,其上的糖链作为生物信息分子参与了细胞生物几乎所有的生物学过程,如细胞识别、粘附与融合、信号转导、免疫与应答等方面都与糖链有关。糖蛋白糖链结构的测定已成为临床实验室诊断疾病,尤其是肿瘤诊断的一个新的研究热点。
本课题在基于DNA测序仪的荧光糖电泳(DSA-FACE)技术的基础上,以乳腺癌细胞系为材料,比较分析了提取细胞膜蛋白和细胞直接切糖技术两种方法处理细胞后对细胞表面N-糖链检测的影响。结果显示,完整细胞直接切糖法检测到的糖链结构无论从数目、丰度均显著优于提取细胞膜蛋白后切糖的方法,该方法结合DSA-FACE技术,是一种可以实现简单、经济、可靠的高通量细胞表面糖链结构检测方法,适用于体外培养细胞表面N-糖链的研究,并为高通量的分析细胞表面糖链的结构以及深入研究糖链生物学功能提供了新的思路。
利用建立的细胞直接进行切糖方法,分析了乳腺细胞系MCF-10A及5种乳腺癌细胞系BT-549、MDA-MB231、MDA-MB435S、MCF-7、Bcap37的细胞表面N-糖链结构。发现正常乳腺细胞系与5种乳腺癌细胞系之间共同存在2种相同的N-糖链结构,并发现这2个N-糖链的含量在不同细胞系间存在差异。我们通过将每种乳腺细胞系分别与MCF-10A乳腺细胞系相比,寻找到每个乳腺癌细胞系各自的特异性N-糖链结构,这些糖链的发现为研究来源不同的乳腺癌细胞系的生理学和病理状态提供了新的数据。
进一步将乳腺导管癌细胞系BT-549与MCF-10A进行了对比分析,发现相同类型的细胞系的细胞表面N-糖链结构比较接近,并筛选出1号糖峰为乳腺导管癌细胞系的特异性糖链。同时还比较了3种乳腺腺癌细胞系MDA-MB231、MDA-MB435S、MCF-7与MCF-10A之间的N-糖链差异,发现相对于乳腺腺癌细胞系,5号峰是正常乳腺细胞系的特异性糖链。同时通过对处于不同临床分期的3种乳腺腺癌细胞系内部之间的比较发现差异较大,其中3号峰、6号峰仅在具有转移特性的乳腺腺癌细胞系中检测到,预示该峰可作为具有转移特性的乳腺腺癌细胞系的特异性糖链。另外通过MCF-10A与乳腺髓样癌细胞系Bcap37比较发现,两者细胞表面N-糖链结构差异显著,其原因可能是由于细胞类型不同而导致这种差异。
本研究建立了结合DSA-FACE技术的细胞表面直接切糖方法,在此基础上,通过对MCF-10A与不同类型的乳腺癌细胞系细胞表面糖链的比较,发现了MCF-10A及乳腺癌细胞系特有的N-糖链,这些糖峰的发现为下一步特异糖链的结构解析奠定了基础,并为糖链的结构与生物学功能的研究提供了新的思路。对于本实验结果的深入研究将有助于阐明细胞表面特征性糖链结构变化规律及乳腺癌的发生、发展和转移的关系。
The cell membrane is the place for information exchanging between cell and surrounding. For the past few years, it was discovered that glucoprotein of cell was the important performer to the function of cell membrane. As the biological information molecular the surface glycans were involved in most biological processes, such as cell recognition, adhesion and fusion, transduction, immunology and response. At present, the detection structure of glycoprotein N-glycans become a determinated method for clinical laboratory diagnosis, especially a hot spot of tumors research.
A new detection technology of human serum glycoprotein N-glycans was established in the research. Two methods were used to detect the surface N-glycans of human breast cancer cell lines based on the DSA-FACE. In one method the glycans were obtained from the membrane protein by detergent extraction. In another they were from the intact cells. The results showed that no matter of the number and abundance of the glycans realeased from the intact cells were greatly superior to those from the membrane protein. It is a more ideal, simple, economical, high-throughput and reliable detection method about surface N-glycans of in vitro culture cell lines. It could provide a new way for analysing of the glycan stucture and biological function.
The N-glycans were detected of which the cell surface of breast cell line MCF-10A and five kinds of breast cancer cell lines BT-549, MDA-MB231, Bcap37, MDA-MB435S, MCF-7 by the new method. There were two N-glycans, of which content was variance, co-exist in all these cell lines. And specific N-glycans of each breast cancer cell line was discoverd by comparing with MCF-10A. ?t provided new data for the research in physiology and pathological of breast cancer cell lines.
Peak 1 was the specific N-glycans of BT-549 cell line by cmparing with MCF-10A. And peak 5 was the specific N-glycans of MCF-10A by cmparing with three breast adenocarcinoma cell.Peak3 and peak 6 might be the specific N-glycans of breast adenocarcinoma which could lead to cancer transferation. Further analysis revealed that there were significant differences between MCF-10A and Bcap37, which can be attributed to different cell types cause such differences.
In this study, we detected the specific surface N-glycans of MCF-10A and the other breast cancer cell lines by the new method.These laid a foundation for the analysis of structural of the specific N-glycans,and provided a new way for the research in structure and biological function of N-glycans.This study would help to clarify the changing of the specific N-glycans in cell surface during the occurrence, development and metastasis of breast cancer.
本课题在基于DNA测序仪的荧光糖电泳(DSA-FACE)技术的基础上,以乳腺癌细胞系为材料,比较分析了提取细胞膜蛋白和细胞直接切糖技术两种方法处理细胞后对细胞表面N-糖链检测的影响。结果显示,完整细胞直接切糖法检测到的糖链结构无论从数目、丰度均显著优于提取细胞膜蛋白后切糖的方法,该方法结合DSA-FACE技术,是一种可以实现简单、经济、可靠的高通量细胞表面糖链结构检测方法,适用于体外培养细胞表面N-糖链的研究,并为高通量的分析细胞表面糖链的结构以及深入研究糖链生物学功能提供了新的思路。
利用建立的细胞直接进行切糖方法,分析了乳腺细胞系MCF-10A及5种乳腺癌细胞系BT-549、MDA-MB231、MDA-MB435S、MCF-7、Bcap37的细胞表面N-糖链结构。发现正常乳腺细胞系与5种乳腺癌细胞系之间共同存在2种相同的N-糖链结构,并发现这2个N-糖链的含量在不同细胞系间存在差异。我们通过将每种乳腺细胞系分别与MCF-10A乳腺细胞系相比,寻找到每个乳腺癌细胞系各自的特异性N-糖链结构,这些糖链的发现为研究来源不同的乳腺癌细胞系的生理学和病理状态提供了新的数据。
进一步将乳腺导管癌细胞系BT-549与MCF-10A进行了对比分析,发现相同类型的细胞系的细胞表面N-糖链结构比较接近,并筛选出1号糖峰为乳腺导管癌细胞系的特异性糖链。同时还比较了3种乳腺腺癌细胞系MDA-MB231、MDA-MB435S、MCF-7与MCF-10A之间的N-糖链差异,发现相对于乳腺腺癌细胞系,5号峰是正常乳腺细胞系的特异性糖链。同时通过对处于不同临床分期的3种乳腺腺癌细胞系内部之间的比较发现差异较大,其中3号峰、6号峰仅在具有转移特性的乳腺腺癌细胞系中检测到,预示该峰可作为具有转移特性的乳腺腺癌细胞系的特异性糖链。另外通过MCF-10A与乳腺髓样癌细胞系Bcap37比较发现,两者细胞表面N-糖链结构差异显著,其原因可能是由于细胞类型不同而导致这种差异。
本研究建立了结合DSA-FACE技术的细胞表面直接切糖方法,在此基础上,通过对MCF-10A与不同类型的乳腺癌细胞系细胞表面糖链的比较,发现了MCF-10A及乳腺癌细胞系特有的N-糖链,这些糖峰的发现为下一步特异糖链的结构解析奠定了基础,并为糖链的结构与生物学功能的研究提供了新的思路。对于本实验结果的深入研究将有助于阐明细胞表面特征性糖链结构变化规律及乳腺癌的发生、发展和转移的关系。
The cell membrane is the place for information exchanging between cell and surrounding. For the past few years, it was discovered that glucoprotein of cell was the important performer to the function of cell membrane. As the biological information molecular the surface glycans were involved in most biological processes, such as cell recognition, adhesion and fusion, transduction, immunology and response. At present, the detection structure of glycoprotein N-glycans become a determinated method for clinical laboratory diagnosis, especially a hot spot of tumors research.
A new detection technology of human serum glycoprotein N-glycans was established in the research. Two methods were used to detect the surface N-glycans of human breast cancer cell lines based on the DSA-FACE. In one method the glycans were obtained from the membrane protein by detergent extraction. In another they were from the intact cells. The results showed that no matter of the number and abundance of the glycans realeased from the intact cells were greatly superior to those from the membrane protein. It is a more ideal, simple, economical, high-throughput and reliable detection method about surface N-glycans of in vitro culture cell lines. It could provide a new way for analysing of the glycan stucture and biological function.
The N-glycans were detected of which the cell surface of breast cell line MCF-10A and five kinds of breast cancer cell lines BT-549, MDA-MB231, Bcap37, MDA-MB435S, MCF-7 by the new method. There were two N-glycans, of which content was variance, co-exist in all these cell lines. And specific N-glycans of each breast cancer cell line was discoverd by comparing with MCF-10A. ?t provided new data for the research in physiology and pathological of breast cancer cell lines.
Peak 1 was the specific N-glycans of BT-549 cell line by cmparing with MCF-10A. And peak 5 was the specific N-glycans of MCF-10A by cmparing with three breast adenocarcinoma cell.Peak3 and peak 6 might be the specific N-glycans of breast adenocarcinoma which could lead to cancer transferation. Further analysis revealed that there were significant differences between MCF-10A and Bcap37, which can be attributed to different cell types cause such differences.
In this study, we detected the specific surface N-glycans of MCF-10A and the other breast cancer cell lines by the new method.These laid a foundation for the analysis of structural of the specific N-glycans,and provided a new way for the research in structure and biological function of N-glycans.This study would help to clarify the changing of the specific N-glycans in cell surface during the occurrence, development and metastasis of breast cancer.
引文
1.孙兴权,李静,耿美玉等.乳腺癌相关糖复合物糖链研究进展.中国药理学通报. 2007, 23(4):425~427
2.金城.糖生物学:基因组学和蛋白质组学的延伸.世界科技研究与发展. 2001, 23(2):31~34
3.李涛,姜颖,贺福初.细胞质膜蛋白质组学.生命的化学. 2002, 6(5): 402~405
4. P.A. Nielsen, J.V. Olsen, A.V. Podtelejnikov, et al. Proteomic mapping of brain plasma membrane proteins. Molecular Cell Proteomics. 2005, 4(4):402~408
5. H. Robert, L.G. Brenda, M.S. Kenneth, et a1. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two dimensional gel electrnphoresis and mass spectrometry. Proteomics. 2002, 2:1479~1488
6. M.S. Margaret, M.R. Beat. Sample preparation for two-dimensional gel electrophoresis. Proteomics. 2003, 3:1408~1417
7.颜建华,去污剂与膜蛋白的提取,国外医学临床生物化学与检验学分册. 1993, 4(14):162~164
8. A. Varki, R. Cummings, J. Esko, et al. Essential of glycobiology. Cold Spring Harbor Laboratory Press. 1999:483~491
9. T. Feizi, B. Mulloy. Carbohydrates and glycoconjugates. Glycomics: the new era of carbohydrate biology. Current Opinion in Structural Biology. 2003, 13(5):602~604
10. D.H. Dube, C.R. Bertozzi. Glycans in cancer and inflammation-potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 2005, 4(6):477~488
11.刘义辉.胸腺细胞表面岩藻糖化抗原在凋亡过程中变化的初步研究.生物化学杂志. 1996, 12(5):559~563
12. D.M. Ratner, E.W. Adams, M.D. Disney, et al. Tools for glycomics: mapping interactions of carbohydrates in biological systems. Chembiochem. 2004, 5(10):1375~1383
13.王克夷.糖类研究的进展和前沿.生命的化学. 1994, 14(5):4~8
14. A. Litynska, M. Prybylo, E. Pochec, et al. Comparison of the lectin-binding pattern in different human melanoma cell lines. Melanoma Res. 2001, 11(3):205~212
15. F.L. Chan, H.L. Choi, S.M. Ho. Analysis of glycoconjugate patterns of normal and hormone-induced dysplastic Noble rat prostate, and an androgen-independentNoble rat prostate tumor, by lectin histochemistry and protein blotting. Prostate. 2001, 46(1):21~32
16. J. Hirabayashi, T. Hashidate, Y. Arata, et al. Biochimica et Biophysica Acta. General Subjects. 2006, 1760(4):616~635
17. B.P. Rempel, H.C. Winter, I.J. Goldstein, et al. Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry. Glycoconjugate. 2003, 19:175~180
18. A. Kameyama, N. Kikuchi, S. Nakaya, et al. A strategy for identification of oligosaccharide structures using observational multistage mass spectral library. Anal Chem. 2005, 77(15):4719~4725
19. M. Froesch, L. M. Bindila, G. Baykut, et al. Coupling of fully automated chip electrospray to Fourier transformation cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing. Rapid Commun Mass Spectrom. 2004, 18(24):3084~3092
20. N. Callewaert, S. Geysens, F. Molemans, et al. Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology. 2001, 11(4):275~281
21. N. Callewaert, H. Van Vlierberghe, A. Van Hecke, et al. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat. Med. 2004, 10(4):429~434
22. V. Vanhooren, X.E. Liu, C. Franceschi et a1. N-glycan profiles as tools in diagnosis of hepatocellular carcinoma and prediction of healthy human ageing. Mechanisms of Ageing and Development. 2009, 130:92~97
23. H. Kogelberg, V.E. Piskarev, Y. Zhang, et al. Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core. Eur J Biochem. 2004, 271(6):1172~1186
24. H.D. Soule, C. McGrath. A simplified method for passage and longterm growth of human mammary cells. In Vitro Cell. Dev. Biol. 1986, 22:6~12
25. S. Hakomori. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv. Exp. Med. Biol. 2001, 491:369~402
26. S. Hakomori. Glycosylation defining cancer malignancy: new wine in an old 27 bottle. Proc. Natl. Acad. Sci. U. S. A. 2002, 99:10231~10233
27. M.A. Hollingsworth, B.J. Swanson. Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer. 2004, 4:45~60
28. A. Stancakova, J. Vajo. The relationship of serum hexuronic acid andmonosaccharide levels to urinary free cortisol and cortisone excretion in healthy subjects and in patients with diabetes mellitus and atherosclerotic peripheral vascular disease. Endokrinologie. 1981, 77:197~205
29. C. Ohyama, S. Orikasa, S. Kawamura, et al. Galactosylgloboside expression in seminoma: Inverse correlation with metastatic potential. Cancer. 1995, 76(7):1043~1050
30.陈智周,范振符,杨剑,等.肿瘤标志物CA153的免疫放射分析及其临床应用.中华肿瘤杂志. 1998, 20(2):1~5
31.刘亚萍,马南花,陆雪秋.乳腺癌患者血清肿瘤标志物检测的临床价值,吉林医学. 2008, 29(15):1235~1237
32.陆云,向俾庭,曾健,等.血清TSGF,CA153、CA125及CEA联合检测对乳腺癌的诊断价值.广西医科大学学报. 2006, 23(2):173~176
33.孟冬娅,胡晓芳,高洪福,等.检测血清VEGF, CEA, TSGF在肿瘤中的临床意义.中国现代医学杂志. 2004, 14(1):117~121
34. J. Wojtacki, W.J. Kruszewski, M. Sliwiska, et a1. Elevatio of serum CA153 antigen: an early indicator of distant metastasis from breast cancer. Retrospective analysis of 733 cases. Przegl Lek. 2001, 58(6):4981~4985
2.金城.糖生物学:基因组学和蛋白质组学的延伸.世界科技研究与发展. 2001, 23(2):31~34
3.李涛,姜颖,贺福初.细胞质膜蛋白质组学.生命的化学. 2002, 6(5): 402~405
4. P.A. Nielsen, J.V. Olsen, A.V. Podtelejnikov, et al. Proteomic mapping of brain plasma membrane proteins. Molecular Cell Proteomics. 2005, 4(4):402~408
5. H. Robert, L.G. Brenda, M.S. Kenneth, et a1. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two dimensional gel electrnphoresis and mass spectrometry. Proteomics. 2002, 2:1479~1488
6. M.S. Margaret, M.R. Beat. Sample preparation for two-dimensional gel electrophoresis. Proteomics. 2003, 3:1408~1417
7.颜建华,去污剂与膜蛋白的提取,国外医学临床生物化学与检验学分册. 1993, 4(14):162~164
8. A. Varki, R. Cummings, J. Esko, et al. Essential of glycobiology. Cold Spring Harbor Laboratory Press. 1999:483~491
9. T. Feizi, B. Mulloy. Carbohydrates and glycoconjugates. Glycomics: the new era of carbohydrate biology. Current Opinion in Structural Biology. 2003, 13(5):602~604
10. D.H. Dube, C.R. Bertozzi. Glycans in cancer and inflammation-potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 2005, 4(6):477~488
11.刘义辉.胸腺细胞表面岩藻糖化抗原在凋亡过程中变化的初步研究.生物化学杂志. 1996, 12(5):559~563
12. D.M. Ratner, E.W. Adams, M.D. Disney, et al. Tools for glycomics: mapping interactions of carbohydrates in biological systems. Chembiochem. 2004, 5(10):1375~1383
13.王克夷.糖类研究的进展和前沿.生命的化学. 1994, 14(5):4~8
14. A. Litynska, M. Prybylo, E. Pochec, et al. Comparison of the lectin-binding pattern in different human melanoma cell lines. Melanoma Res. 2001, 11(3):205~212
15. F.L. Chan, H.L. Choi, S.M. Ho. Analysis of glycoconjugate patterns of normal and hormone-induced dysplastic Noble rat prostate, and an androgen-independentNoble rat prostate tumor, by lectin histochemistry and protein blotting. Prostate. 2001, 46(1):21~32
16. J. Hirabayashi, T. Hashidate, Y. Arata, et al. Biochimica et Biophysica Acta. General Subjects. 2006, 1760(4):616~635
17. B.P. Rempel, H.C. Winter, I.J. Goldstein, et al. Characterization of the recognition of blood group B trisaccharide derivatives by the lectin from Marasmius oreades using frontal affinity chromatography-mass spectrometry. Glycoconjugate. 2003, 19:175~180
18. A. Kameyama, N. Kikuchi, S. Nakaya, et al. A strategy for identification of oligosaccharide structures using observational multistage mass spectral library. Anal Chem. 2005, 77(15):4719~4725
19. M. Froesch, L. M. Bindila, G. Baykut, et al. Coupling of fully automated chip electrospray to Fourier transformation cyclotron resonance mass spectrometry for high-performance glycoscreening and sequencing. Rapid Commun Mass Spectrom. 2004, 18(24):3084~3092
20. N. Callewaert, S. Geysens, F. Molemans, et al. Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology. 2001, 11(4):275~281
21. N. Callewaert, H. Van Vlierberghe, A. Van Hecke, et al. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat. Med. 2004, 10(4):429~434
22. V. Vanhooren, X.E. Liu, C. Franceschi et a1. N-glycan profiles as tools in diagnosis of hepatocellular carcinoma and prediction of healthy human ageing. Mechanisms of Ageing and Development. 2009, 130:92~97
23. H. Kogelberg, V.E. Piskarev, Y. Zhang, et al. Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core. Eur J Biochem. 2004, 271(6):1172~1186
24. H.D. Soule, C. McGrath. A simplified method for passage and longterm growth of human mammary cells. In Vitro Cell. Dev. Biol. 1986, 22:6~12
25. S. Hakomori. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv. Exp. Med. Biol. 2001, 491:369~402
26. S. Hakomori. Glycosylation defining cancer malignancy: new wine in an old 27 bottle. Proc. Natl. Acad. Sci. U. S. A. 2002, 99:10231~10233
27. M.A. Hollingsworth, B.J. Swanson. Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer. 2004, 4:45~60
28. A. Stancakova, J. Vajo. The relationship of serum hexuronic acid andmonosaccharide levels to urinary free cortisol and cortisone excretion in healthy subjects and in patients with diabetes mellitus and atherosclerotic peripheral vascular disease. Endokrinologie. 1981, 77:197~205
29. C. Ohyama, S. Orikasa, S. Kawamura, et al. Galactosylgloboside expression in seminoma: Inverse correlation with metastatic potential. Cancer. 1995, 76(7):1043~1050
30.陈智周,范振符,杨剑,等.肿瘤标志物CA153的免疫放射分析及其临床应用.中华肿瘤杂志. 1998, 20(2):1~5
31.刘亚萍,马南花,陆雪秋.乳腺癌患者血清肿瘤标志物检测的临床价值,吉林医学. 2008, 29(15):1235~1237
32.陆云,向俾庭,曾健,等.血清TSGF,CA153、CA125及CEA联合检测对乳腺癌的诊断价值.广西医科大学学报. 2006, 23(2):173~176
33.孟冬娅,胡晓芳,高洪福,等.检测血清VEGF, CEA, TSGF在肿瘤中的临床意义.中国现代医学杂志. 2004, 14(1):117~121
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