生物活性短肽RGD的合成及其在PET表面接枝方法的研究
|
摘要
生物材料在人类的疾病治疗与康复中起着重要的作用。心血管系统的疾病对生物材料的要求较高。传统的人造心血管材料在应用中不同程度发生过凝血、补体激活等反应，直接或间接地导致血栓的形成，最终导致植入物的失败甚至产生并发症危及患者的生命。因此，改善心血管材料的血液相容性，一直是生物材料研究的重要课题。
RGD是大多数细胞表面粘附分子能识别的配体上的最小氨基酸序列，将RGD序列结合到材料表面上，来自细胞膜表面的整合素(Integrin)与材料表面的RGD通过配体一受体的结合，能在一定程度上促进细胞在材料表面的粘附、铺展、生长，最终融合成层。实现在材料表面形成具生物活性的细胞层，提高生物材料血液相容性。
本论文是国家自然科学基金项目：“高分子材料表面细胞可识别分子模型及内皮化机理的研究”的一部分，即在高分子材料表面共价引入人工合成的短肽RGD以达到让内皮细胞与之特异结合并得到更加牢固内皮化表面的目的。
实验进行了甘氨酸、天冬氨酸、精氨酸三种氨基酸的保护；RGD三肽的液相合成与纯化；活性基团-COOH的引入和RGD的接枝；材料的内皮细胞生长实验的研究等。对保护的氨基酸产物用薄层色谱(TLC)进行了反应进程的跟踪及产物纯度的初步鉴定。结合熔点测试、旋光分析及红外等手段对合成产物进行了定性分析。用柱层析的方法对RGD进行纯化，用TLC相对R_f值与美国SIGMA公司标样对照以确认产物。光电子能谱对以羧基为活性基团的接肽反应结果进行分析。光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。
实验得到了叔丁氧羰基(Boc)保护的甘氨酸、苄酯(Bzl)保护的天冬氨酸、硝基(NO_2)保护的精氨酸三种产物并液相合成了RGD三肽，紫外辐照法将活性基团-COOH接到材料的表面，内皮细胞生长实验结果表明，具有生物活性RGD序列已成功接枝到材料表面，并对材料内皮细胞种植起到了促进作用。
建议下一步的工作，应在现阶段初步成功的实验基础上，进行细胞生长情况与RGD或含RGD的更长短肽接枝量的关系，以及在流体力学条件下比较接枝与否与内皮化牢固性、持久性的关系等，从而对内皮化的机理进一步深入研究。
Biomaterials play an important role in human disease-treatment and healing. Diseases in cardiovascular system have higher requirement about biomaterials. In the application, traditional artificial cardiovascular materials have blood coagulation, alexin-activation and other effects in different degree which result the formation of thrombus directly or indirectly, and finally lead to the failure of implantation materials or even cause complication and endanger the patients' lives. Therefore it has been an important subject in the materials study to improve the blood compatability of cardiovascular materials.
RGD is the minimum ammonia serial on the ligand, which can be recognized by most of the integrins. Connect the RGD serial on the materials surface, integrin, which comes from membrane, one of the cell adhesion molecule (CAM) on the cell surface, combines with RGD on the materials surface through ligand-acceptor. It can to some extent help the cell adhesion, spreads, grow, and finally mix to layers on the materials surface. Cell layers with biological activities form on the materials surface which can promote the blood compatibility of the biomaterials.
This article is the part of "Recognizable Molecular Mode on the Polymer Materials Surface Cell and Study on the mechanism of Endothelialization."which is the Nature Science Fund project. That is covalence induce artificial combine RGD on the polymer materials surface to make endothelial cells to combine with its speciality, so that more fixed endothelializate surface.
TLC method was adopted to trace the reaction process and primary test the products purity on the protected ammonia. Analysis over the combined products was carried out with the method of the testing of melting point, polarimeter and IR. RGD was purified with column chromatography. TLC XPS were used to analysis the
results. Optical microscope and SEM observe the status of the endothelial cells growth.
Protection over Arg, Gly, Asp were carried and in the experiment Liquid phase synthesis and purification of RGB were taken. Protecting results from Gly protected by Boc, Asp protected by Bzl, Arg by N02 were obtained in the experiment. Pure RGD was obtained in the liquid phase synthesis. Active group -COOH was connected on the materials surface by UV radiation. The endothelialization result shows that RGD serial is successfully connected on the materials surface and improve the effect of materials endothelialization.
In the future the study should be focus on the quantitation of cell growth and grafting of RGD, and the grafting of peptides longer than RGD such as RGDX following the way brought forward , and the relation of grafting and endothelialization on the condition of hydrodynamics.
RGD是大多数细胞表面粘附分子能识别的配体上的最小氨基酸序列，将RGD序列结合到材料表面上，来自细胞膜表面的整合素(Integrin)与材料表面的RGD通过配体一受体的结合，能在一定程度上促进细胞在材料表面的粘附、铺展、生长，最终融合成层。实现在材料表面形成具生物活性的细胞层，提高生物材料血液相容性。
本论文是国家自然科学基金项目：“高分子材料表面细胞可识别分子模型及内皮化机理的研究”的一部分，即在高分子材料表面共价引入人工合成的短肽RGD以达到让内皮细胞与之特异结合并得到更加牢固内皮化表面的目的。
实验进行了甘氨酸、天冬氨酸、精氨酸三种氨基酸的保护；RGD三肽的液相合成与纯化；活性基团-COOH的引入和RGD的接枝；材料的内皮细胞生长实验的研究等。对保护的氨基酸产物用薄层色谱(TLC)进行了反应进程的跟踪及产物纯度的初步鉴定。结合熔点测试、旋光分析及红外等手段对合成产物进行了定性分析。用柱层析的方法对RGD进行纯化，用TLC相对R_f值与美国SIGMA公司标样对照以确认产物。光电子能谱对以羧基为活性基团的接肽反应结果进行分析。光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。
实验得到了叔丁氧羰基(Boc)保护的甘氨酸、苄酯(Bzl)保护的天冬氨酸、硝基(NO_2)保护的精氨酸三种产物并液相合成了RGD三肽，紫外辐照法将活性基团-COOH接到材料的表面，内皮细胞生长实验结果表明，具有生物活性RGD序列已成功接枝到材料表面，并对材料内皮细胞种植起到了促进作用。
建议下一步的工作，应在现阶段初步成功的实验基础上，进行细胞生长情况与RGD或含RGD的更长短肽接枝量的关系，以及在流体力学条件下比较接枝与否与内皮化牢固性、持久性的关系等，从而对内皮化的机理进一步深入研究。
Biomaterials play an important role in human disease-treatment and healing. Diseases in cardiovascular system have higher requirement about biomaterials. In the application, traditional artificial cardiovascular materials have blood coagulation, alexin-activation and other effects in different degree which result the formation of thrombus directly or indirectly, and finally lead to the failure of implantation materials or even cause complication and endanger the patients' lives. Therefore it has been an important subject in the materials study to improve the blood compatability of cardiovascular materials.
RGD is the minimum ammonia serial on the ligand, which can be recognized by most of the integrins. Connect the RGD serial on the materials surface, integrin, which comes from membrane, one of the cell adhesion molecule (CAM) on the cell surface, combines with RGD on the materials surface through ligand-acceptor. It can to some extent help the cell adhesion, spreads, grow, and finally mix to layers on the materials surface. Cell layers with biological activities form on the materials surface which can promote the blood compatibility of the biomaterials.
This article is the part of "Recognizable Molecular Mode on the Polymer Materials Surface Cell and Study on the mechanism of Endothelialization."which is the Nature Science Fund project. That is covalence induce artificial combine RGD on the polymer materials surface to make endothelial cells to combine with its speciality, so that more fixed endothelializate surface.
TLC method was adopted to trace the reaction process and primary test the products purity on the protected ammonia. Analysis over the combined products was carried out with the method of the testing of melting point, polarimeter and IR. RGD was purified with column chromatography. TLC XPS were used to analysis the
results. Optical microscope and SEM observe the status of the endothelial cells growth.
Protection over Arg, Gly, Asp were carried and in the experiment Liquid phase synthesis and purification of RGB were taken. Protecting results from Gly protected by Boc, Asp protected by Bzl, Arg by N02 were obtained in the experiment. Pure RGD was obtained in the liquid phase synthesis. Active group -COOH was connected on the materials surface by UV radiation. The endothelialization result shows that RGD serial is successfully connected on the materials surface and improve the effect of materials endothelialization.
In the future the study should be focus on the quantitation of cell growth and grafting of RGD, and the grafting of peptides longer than RGD such as RGDX following the way brought forward , and the relation of grafting and endothelialization on the condition of hydrodynamics.
引文
1. DR Absolom, Endothelialization of Polymer Surfaces, J. Biomed. Mater. Res. 1988, 22: 271～279
2. PB Van Wachem, Interaction of Ualtured Human Endothelial Cells with Polymeric Surface of Different Wettabilities, Biomaterials, 1985, 6(11): 403～410
3. X.F.Walboomers, H.J.E.Croes, L. A. Ginsel, et. al, Growth Behavior of Fibroblasts on Various Microgrooved Implant Materials, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI, U.S.A
4. Zbigniew. Gugala, Sylmester. Gogoleaski, Growth Differentiation and Activity of Rat Marrow Stromal Cells on Resorbable Poly(L/DL-Lactide) Membranes with Defined Surface Characteristic, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
5. Marion P. Ovilieri, Katherine S. Tweden, Human serum albumin and fibrinogen interaction with adsorbed RGD-containing peptide, J. Biomed. Mater. Res, 1999, 46, 355～359
6. Geoffrey D.Moodie, Diana. M.Ferris, Bone-Derived Cell Spreading on and Stability of RGD-Coated Substrates, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Trasaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
7. Martin C. Peters, Lonnie D. Shea, David J. Mooney, Development of VEGF Releasing Three Dimension Matrices for Cells Transplantation, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
8．魏蜀亮，粘附蛋白在环氧交联牛心包心瓣膜材料内皮化中的作用，华西医科大学研究生学位论文，1996年5月
9．周序龙，邓漪平，碱性成纤维细胞生长因子对内皮细胞在聚氨酯材料表面粘附的影响，生物医学工程杂志，1998：15(3)：214～217
10. Yoshiaki Hirano, Yoshihiro Kando and Toshino Hayashi et al, Synthesis and Cell Attachment Activity of Bioactive Oligopeptide: RGD, RGDS, RGDV, and RGDT, J. Biomed. Mater. Res. 1991, 25, 1523～1534,
11. M.C.Porte-Durrieu, C.Labrugere, F.Villars, et al, Development of RGD peptide grafted onto silica surfaces: XPS characterization and human endothelial cell interaction, J. Biomed. Mater. Res. 1999, 46, 368～375,
12．张开，高分子界面科学，中国石化出版社，1997．3，
13．朱如瑾，张开，化学与粘合，1990(4)：234
14. Y. Nakao;H.Dmichi,j.Appl,Polym, Sci., 1984,29:1041
15. G.Odian, J.Polym, Chem, ed.,1984;22: 769
16. J.K. Pieke,et.al.,Polym. Sci., 1963,C1:117
17．中尾一宗，日本接着协会志，1965，1(3)：7
18. M. Suzuki, A. Kishida, H. Iwata, and Y. Ikada, Graft copolymerization of acrylamide onto a polyethylene surface protreated with a glow dicharge, Macromoleculars, 1986, 19(7): 1345～1356
19. R. Sipehia, and A. S. Chawla, Albuminated polymer surfaces for biomedical application, Biomat. Med. Dev., Art. Org., 1982, 10(4): 229～246,
20. Hans J. Griesser, Jonathan H. Hodgkin and Robert Schmidt, Plasma techniques for production of permanent hydrophilic polymer surfaces for biomedical applications, Progress in Biomedical Polymers, Plenum Press, New York, 1990
21．周成飞，医用高分子表面等离子体改性的研究动向，生物医学工程杂志，1989，6(3)：211～215
22. R.A.Bragole,Adhesive Age, 1974,17(4):24
23. B.P.760,611,(1957)
24. L.E.Wolinski,U.S.P.3.801,446(1957)
25. Niklas,Ger.P.,1,106,069(1961)
26. M.Hudis,L.E.PresCott,J.Polym, Sci., 1972,B10,179
27. M.Hudis,J.Appl. Polym, Sci., 1972,16,2397
28．筏羲人著，许德恒等译，高分子表面的基础和应用，化学工业出版社，1990
29. Sofukku S,Hoshida A,Shimada T.Synthesis of cyclic oligopeptides containing an Arg-Gly-Asp sequence.Chemical &Pharmaceutical Bulletin 1999,47:(12) 1799-1801
30．赵明，彭师奇，迪丽努尔。含RGD四肽的合成与功能。药学学报，1997，32(4)：271-277．
31. H.Marlon Zhong,Michael N.Solid-phase sythesis of Arginine-containing peptides by guanidine attachment to a sulfonyl linker. J. Org. Chem. 1997, 62,9326-9330
32. Maynard HD, Okada SY, Grubbs RH. Macromlecules.2000,33(17);6239-6248.
33．陈育新，张学忠，陈松明，吴晓霞。在AOT/异辛烷反相胶束体系中酶法合成RGD前体二肽。中国生物化学与分子生物学报，1999，15(2)：148-150。
34. So JE,Shin JS,Kim BG.Protease-catalyzed tripeptide(RGD)sythesis.Enzyme and Microbial Technology,2000,26: (2-4) 108-114.
35. Y. Uyama and Y. Ikada Graft polymerization of acrylamide onto UV-irradiated films, J. Appl. Poly. Sci., 1988, Vol. 36, 1087～1096
36. Yoshitaka Ogiwara, Masaru Takumi and Hitoshi Kubota, Photoinduced grafting of acylamide onto polyethylene film by means of two-step method, J. Appl. Poly. Sci., 1982, Vol.27, 3743～3750
37. Zhang Dei Yao and Bengt Ranby, Surface modification by continuous graft copolymerization. I. Photoinitiated graft copolymerization onto polyethylene tape film surface, J. Appl. Poly. Sci., 1990, Vol.40: 1647～1661
38. Emiko Uchida, Yoshikimi Uyama, Surface graft polymerization of acrylamide onto PET film by UV irradiation, J. Appl. Poly. Sci., Part A: polymer chemistry, 1989, Vol.27, 527～537
39. Emiko Uchida, Yoshikimi Uyama, A novel method for graft polymerization onto PET film surface by irradiation without degassing, J. Appl. Poly. Sci., 1990, Vol.41, 677～687,
40. Wantai Yang and Bengt Ranby, Bulk surface photografting process and its applications. I. Reaction and Kinetics, J. Appl. Poly. Sci., 1996, Vol.62, 533～543,
41．黄惟德，陈常庆著，多肽合成，科学出版社，1985
42. Hofmann K., Peekham, W.D., and Rheimer, A.J., Studied on Polypeptides. Ⅶ The Synthesis of Peptides Containing Arginine, J. Am. Chem. Soc. 1957, 22, 1515～1522
43．杨锦宗编著，工业有机合成基础，中国石化出版社，1998．12
44. D.F. Detar, R. Silverstein,J.Am.Chem.Soc.,88,1013,1966
45. J.Kovacs,L.Kisfaludy,M.Q.Ceprini,J.Am.Chen.Soc.,89,183,1967
46. R.B.Merrifield, B.F.Gisin,A.Bodanszky, J.Org. Chem.,40,1570,1975
47. H.Gross,L.Kilk, Tetreahedron,24,6935,1968
48. M.Fujino et al.,Chem. Pharm. Bull.22,1875,1974
49. W. koening,R.Geiger, Chem. Ber., 103,788,1970
50. W. koening,R.Geiger, Chem. Ber., 103,2024,1970
51. W. koening,R.Geiger, Chem. Ber., 103,2034,1970
52．谭佩幸，陶宗晋，祁国荣，徐俊杰，现代化学试剂手册，第三分册，生化试剂(一)，化学工业出版社
53．大森英三，功能性丙烯酸树脂，化学工业出版社，1993．12
54．C.R.洛著，亲和色谱导论，1979
55. Greg T. Hermanson, A Krishna Malla and Paul K. Smith, Immobi;ized Affinity Ligand Techniques, Academic Press, 1997
56．蒋中华，张建辉，生物的固定化技术及应用，化学工业出版社，1998
57. Staros, J. V.,Wright,R.W., and Swingle,D.M.Anal.Biochem.156,220.1986
58. Grabarek, Z.,and Gergely, J.Anal. Biochem. 185,131, 1990
59. Hoare,D.G., and Koshland,D.E.J.Amer. Chem. Soc.88,2057,1966
60. Hoare,D.G., and Koshland,D.E.J.Amer. Chem. Soc.242,2447,1967
61．司徒镇强，吴军正主编，细胞培养，世界图书出版社，1996．4
2. PB Van Wachem, Interaction of Ualtured Human Endothelial Cells with Polymeric Surface of Different Wettabilities, Biomaterials, 1985, 6(11): 403～410
3. X.F.Walboomers, H.J.E.Croes, L. A. Ginsel, et. al, Growth Behavior of Fibroblasts on Various Microgrooved Implant Materials, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI, U.S.A
4. Zbigniew. Gugala, Sylmester. Gogoleaski, Growth Differentiation and Activity of Rat Marrow Stromal Cells on Resorbable Poly(L/DL-Lactide) Membranes with Defined Surface Characteristic, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
5. Marion P. Ovilieri, Katherine S. Tweden, Human serum albumin and fibrinogen interaction with adsorbed RGD-containing peptide, J. Biomed. Mater. Res, 1999, 46, 355～359
6. Geoffrey D.Moodie, Diana. M.Ferris, Bone-Derived Cell Spreading on and Stability of RGD-Coated Substrates, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Trasaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
7. Martin C. Peters, Lonnie D. Shea, David J. Mooney, Development of VEGF Releasing Three Dimension Matrices for Cells Transplantation, 1999 SOCIETY FOR BIOMATERIALS 25th Annual Meeting Transaction, April 28 May 2. 1999 Rhode Island Convention Center Providene, RI,U.S.A
8．魏蜀亮，粘附蛋白在环氧交联牛心包心瓣膜材料内皮化中的作用，华西医科大学研究生学位论文，1996年5月
9．周序龙，邓漪平，碱性成纤维细胞生长因子对内皮细胞在聚氨酯材料表面粘附的影响，生物医学工程杂志，1998：15(3)：214～217
10. Yoshiaki Hirano, Yoshihiro Kando and Toshino Hayashi et al, Synthesis and Cell Attachment Activity of Bioactive Oligopeptide: RGD, RGDS, RGDV, and RGDT, J. Biomed. Mater. Res. 1991, 25, 1523～1534,
11. M.C.Porte-Durrieu, C.Labrugere, F.Villars, et al, Development of RGD peptide grafted onto silica surfaces: XPS characterization and human endothelial cell interaction, J. Biomed. Mater. Res. 1999, 46, 368～375,
12．张开，高分子界面科学，中国石化出版社，1997．3，
13．朱如瑾，张开，化学与粘合，1990(4)：234
14. Y. Nakao;H.Dmichi,j.Appl,Polym, Sci., 1984,29:1041
15. G.Odian, J.Polym, Chem, ed.,1984;22: 769
16. J.K. Pieke,et.al.,Polym. Sci., 1963,C1:117
17．中尾一宗，日本接着协会志，1965，1(3)：7
18. M. Suzuki, A. Kishida, H. Iwata, and Y. Ikada, Graft copolymerization of acrylamide onto a polyethylene surface protreated with a glow dicharge, Macromoleculars, 1986, 19(7): 1345～1356
19. R. Sipehia, and A. S. Chawla, Albuminated polymer surfaces for biomedical application, Biomat. Med. Dev., Art. Org., 1982, 10(4): 229～246,
20. Hans J. Griesser, Jonathan H. Hodgkin and Robert Schmidt, Plasma techniques for production of permanent hydrophilic polymer surfaces for biomedical applications, Progress in Biomedical Polymers, Plenum Press, New York, 1990
21．周成飞，医用高分子表面等离子体改性的研究动向，生物医学工程杂志，1989，6(3)：211～215
22. R.A.Bragole,Adhesive Age, 1974,17(4):24
23. B.P.760,611,(1957)
24. L.E.Wolinski,U.S.P.3.801,446(1957)
25. Niklas,Ger.P.,1,106,069(1961)
26. M.Hudis,L.E.PresCott,J.Polym, Sci., 1972,B10,179
27. M.Hudis,J.Appl. Polym, Sci., 1972,16,2397
28．筏羲人著，许德恒等译，高分子表面的基础和应用，化学工业出版社，1990
29. Sofukku S,Hoshida A,Shimada T.Synthesis of cyclic oligopeptides containing an Arg-Gly-Asp sequence.Chemical &Pharmaceutical Bulletin 1999,47:(12) 1799-1801
30．赵明，彭师奇，迪丽努尔。含RGD四肽的合成与功能。药学学报，1997，32(4)：271-277．
31. H.Marlon Zhong,Michael N.Solid-phase sythesis of Arginine-containing peptides by guanidine attachment to a sulfonyl linker. J. Org. Chem. 1997, 62,9326-9330
32. Maynard HD, Okada SY, Grubbs RH. Macromlecules.2000,33(17);6239-6248.
33．陈育新，张学忠，陈松明，吴晓霞。在AOT/异辛烷反相胶束体系中酶法合成RGD前体二肽。中国生物化学与分子生物学报，1999，15(2)：148-150。
34. So JE,Shin JS,Kim BG.Protease-catalyzed tripeptide(RGD)sythesis.Enzyme and Microbial Technology,2000,26: (2-4) 108-114.
35. Y. Uyama and Y. Ikada Graft polymerization of acrylamide onto UV-irradiated films, J. Appl. Poly. Sci., 1988, Vol. 36, 1087～1096
36. Yoshitaka Ogiwara, Masaru Takumi and Hitoshi Kubota, Photoinduced grafting of acylamide onto polyethylene film by means of two-step method, J. Appl. Poly. Sci., 1982, Vol.27, 3743～3750
37. Zhang Dei Yao and Bengt Ranby, Surface modification by continuous graft copolymerization. I. Photoinitiated graft copolymerization onto polyethylene tape film surface, J. Appl. Poly. Sci., 1990, Vol.40: 1647～1661
38. Emiko Uchida, Yoshikimi Uyama, Surface graft polymerization of acrylamide onto PET film by UV irradiation, J. Appl. Poly. Sci., Part A: polymer chemistry, 1989, Vol.27, 527～537
39. Emiko Uchida, Yoshikimi Uyama, A novel method for graft polymerization onto PET film surface by irradiation without degassing, J. Appl. Poly. Sci., 1990, Vol.41, 677～687,
40. Wantai Yang and Bengt Ranby, Bulk surface photografting process and its applications. I. Reaction and Kinetics, J. Appl. Poly. Sci., 1996, Vol.62, 533～543,
41．黄惟德，陈常庆著，多肽合成，科学出版社，1985
42. Hofmann K., Peekham, W.D., and Rheimer, A.J., Studied on Polypeptides. Ⅶ The Synthesis of Peptides Containing Arginine, J. Am. Chem. Soc. 1957, 22, 1515～1522
43．杨锦宗编著，工业有机合成基础，中国石化出版社，1998．12
44. D.F. Detar, R. Silverstein,J.Am.Chem.Soc.,88,1013,1966
45. J.Kovacs,L.Kisfaludy,M.Q.Ceprini,J.Am.Chen.Soc.,89,183,1967
46. R.B.Merrifield, B.F.Gisin,A.Bodanszky, J.Org. Chem.,40,1570,1975
47. H.Gross,L.Kilk, Tetreahedron,24,6935,1968
48. M.Fujino et al.,Chem. Pharm. Bull.22,1875,1974
49. W. koening,R.Geiger, Chem. Ber., 103,788,1970
50. W. koening,R.Geiger, Chem. Ber., 103,2024,1970
51. W. koening,R.Geiger, Chem. Ber., 103,2034,1970
52．谭佩幸，陶宗晋，祁国荣，徐俊杰，现代化学试剂手册，第三分册，生化试剂(一)，化学工业出版社
53．大森英三，功能性丙烯酸树脂，化学工业出版社，1993．12
54．C.R.洛著，亲和色谱导论，1979
55. Greg T. Hermanson, A Krishna Malla and Paul K. Smith, Immobi;ized Affinity Ligand Techniques, Academic Press, 1997
56．蒋中华，张建辉，生物的固定化技术及应用，化学工业出版社，1998
57. Staros, J. V.,Wright,R.W., and Swingle,D.M.Anal.Biochem.156,220.1986
58. Grabarek, Z.,and Gergely, J.Anal. Biochem. 185,131, 1990
59. Hoare,D.G., and Koshland,D.E.J.Amer. Chem. Soc.88,2057,1966
60. Hoare,D.G., and Koshland,D.E.J.Amer. Chem. Soc.242,2447,1967
61．司徒镇强，吴军正主编，细胞培养，世界图书出版社，1996．4