银杏细胞固定化培养及其银杏内酯类成分的研究
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摘要
本文对银杏细胞固定化培养及其次生代谢产物银杏内酯的产生进行了较为系统的研究。
进行了优良种系的诱导和优选，得到了一种生长快、分散性好，并很好的符合固定化要求的细胞株系；对固定化条件进行了最优化配置--从载体预处理、载体种类、载体量、大小和溶氧到细胞接种量系统的研究；采用均匀设计寻求一种高固定化、高代谢及长期保持细胞活力的激素配比，最终得到一种基本满足上述要求的培养基。本固定化结果明显的提高了用细胞培养方法生产银杏内酯的效率。
The studies on cell immobilization culture of Ginkgo biloba L, as well as the production of their secondary metabolite -Ginkgolides were reported in this paper. A cell line, which fit the request of the immobilization culture very well, was obtained by induction and optimized selection. The cell line grown quickly and well dispersed. The optimized result of cell immobilization cultures was acquired, including the support matrices pretreatment, the class and size of support matrices, the level of dissolved 62 and inoculum. The optimal ratio of hormones was got by uniform design according to its high immobilization level, high metabolism and retaining vigorous cells with long period. A medium which fit well all the conditions was obtained, and the efficiency of producing Ginkgolides by cell culture improved obviously.
进行了优良种系的诱导和优选，得到了一种生长快、分散性好，并很好的符合固定化要求的细胞株系；对固定化条件进行了最优化配置--从载体预处理、载体种类、载体量、大小和溶氧到细胞接种量系统的研究；采用均匀设计寻求一种高固定化、高代谢及长期保持细胞活力的激素配比，最终得到一种基本满足上述要求的培养基。本固定化结果明显的提高了用细胞培养方法生产银杏内酯的效率。
The studies on cell immobilization culture of Ginkgo biloba L, as well as the production of their secondary metabolite -Ginkgolides were reported in this paper. A cell line, which fit the request of the immobilization culture very well, was obtained by induction and optimized selection. The cell line grown quickly and well dispersed. The optimized result of cell immobilization cultures was acquired, including the support matrices pretreatment, the class and size of support matrices, the level of dissolved 62 and inoculum. The optimal ratio of hormones was got by uniform design according to its high immobilization level, high metabolism and retaining vigorous cells with long period. A medium which fit well all the conditions was obtained, and the efficiency of producing Ginkgolides by cell culture improved obviously.
引文
[1] Sticher O，银杏内酯的质量，国外医药·植物药分册，1993，Vol8，No 4
[2] T A VAN BEER. H. A SCHEEREN, T. RANTIO, W Ch, MELGER and Gp. LELYVELD Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and pharmacerticals J. of Chromatogr. 543(1991) , 375-238
[3] 赵鸿莲，银杏组织和细胞培养及其次生代谢产物银杏内酯的研究，[学位论文]，1997
[4] Tulecke W (1957), Amer J Bot 44, 601-608
[5] Carrier D. J., Cosentino G., Neu feld R., et al (1990) Plant Cell Rep. 8, 635-638
[6] Carrier, D. J. Chanret, N. Maneini, M. (1990), Plant cell Rep, 10: 256-259
[7] Endress R., Plant Cell Biotechnlogy , chapter7. Immobilized of plant cell, Berlin:Springer - Verlag Berlin Heidelberg, 1994, 256-268
[8] M. J. C. Rhodes, J. I. Smith, and R. J. Robins Factors affecting the immobilization of plant cells on reticulated polyurethane foam partides. Appl. Microbil. Biotechnol 1987, 26:28-35
[9] 潘祖仁，高分子化学，化学工业出版社，135-149．
[10] Richard J., Polyurethanes in biomedical applications : promises of the past, present realities and vibrant futures: zdrahala, 60 years polyurethanes: [Int. Symb. Exhib] 1998, 304(E). Edited by Kresta, Jiri E.; Eleanore W .T.; Lancaster, Pa.
[11] 聚二甲基硅氧烷／聚氨酯共混体系的表面结构和抗凝血性能，生物医学工程学杂志，1995，12(3)：191
[12] 甘烦远，郑光植，植物培养细胞次级代谢产物高产细胞系的筛选，国外医药·植物药分册，1990，5(1)：10-14
[13] 刘佳佳，李晓如，郭勇，郑穗平 缺氧胁迫法选育高产银杏内酯悬浮细胞系研
究，天然产物研究与开发，13(2)：1-4
[14] 熊宗贵等，生物技术制药，高等教育出版社，2000，181
[15] 袁玉信，微量元素在植物生活中的作用，生物学通报，1996，31(4)，4-8
[16] J. L. Iborra, J. Guardiola, S. Montaner, et. Enhanced accumulation of anthocyanins in vitis vinifera cells immobilized in PUF. Enzyme Microb. Technol. 1994, 16(May): 416-419
[17] 李新明，侯崇生，陈士云等，九连小檗悬浮培养细胞的固定化研究，植物学报，1993，35(增刊)：101-105
[18] Taku Matsushita, Hiromichi Hidaka, Ken-ichi Kamihata, et. High density culture of anchorage-dependent animal cells by PUF packed-bed culture systems. Appl. Microbiol. Biotechnol.1991,35:159-164
[19] Robins RJ, Parr A J, Richards S R, Rhodes M J C (1986)Studies of environmental features of immobilized plant cell. In: Morris P, Scragg AH, Stafford A, Fowler M W (eds) Secondary Metabolism in Plant Cell Cultures, Cambridge University Press, Cambridge, PP162-172
[20] 武汉大学、复旦大学生物系微生物学教研室编，微生物学[第二版]，高等教育出版社
[21] 元英进，胡宗定：聚氨酯泡沫固定化长春花细胞生产生物碱的研究，化学反应工程与工艺，1994，10(2)，195-199
[22] 波钦诺克XH：植物生物化学分析方法，荆家海译，科学出版社，P145
[23] 中华人民共和国药典，中华人民共和国药典委员会，一九九五年版，一部，附录
[24] 郑玉果，采用愈伤组织固体培养法生产银杏内酯的初步研究，[学位论文]，1996
[25] Steinke, B. Müller, B. , Wager, H. , Planta Med. 1993, 59 (2) ,150 -160
[26]王佃亮，肖成祖，动物细胞高密度培养用多孔微球，生物工程进展，1998，18(2)，46-49．
[27] Messing, R.A,et al.Biotechnol. Bioeng., 1979,21,49-58
[2] T A VAN BEER. H. A SCHEEREN, T. RANTIO, W Ch, MELGER and Gp. LELYVELD Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and pharmacerticals J. of Chromatogr. 543(1991) , 375-238
[3] 赵鸿莲，银杏组织和细胞培养及其次生代谢产物银杏内酯的研究，[学位论文]，1997
[4] Tulecke W (1957), Amer J Bot 44, 601-608
[5] Carrier D. J., Cosentino G., Neu feld R., et al (1990) Plant Cell Rep. 8, 635-638
[6] Carrier, D. J. Chanret, N. Maneini, M. (1990), Plant cell Rep, 10: 256-259
[7] Endress R., Plant Cell Biotechnlogy , chapter7. Immobilized of plant cell, Berlin:Springer - Verlag Berlin Heidelberg, 1994, 256-268
[8] M. J. C. Rhodes, J. I. Smith, and R. J. Robins Factors affecting the immobilization of plant cells on reticulated polyurethane foam partides. Appl. Microbil. Biotechnol 1987, 26:28-35
[9] 潘祖仁，高分子化学，化学工业出版社，135-149．
[10] Richard J., Polyurethanes in biomedical applications : promises of the past, present realities and vibrant futures: zdrahala, 60 years polyurethanes: [Int. Symb. Exhib] 1998, 304(E). Edited by Kresta, Jiri E.; Eleanore W .T.; Lancaster, Pa.
[11] 聚二甲基硅氧烷／聚氨酯共混体系的表面结构和抗凝血性能，生物医学工程学杂志，1995，12(3)：191
[12] 甘烦远，郑光植，植物培养细胞次级代谢产物高产细胞系的筛选，国外医药·植物药分册，1990，5(1)：10-14
[13] 刘佳佳，李晓如，郭勇，郑穗平 缺氧胁迫法选育高产银杏内酯悬浮细胞系研
究，天然产物研究与开发，13(2)：1-4
[14] 熊宗贵等，生物技术制药，高等教育出版社，2000，181
[15] 袁玉信，微量元素在植物生活中的作用，生物学通报，1996，31(4)，4-8
[16] J. L. Iborra, J. Guardiola, S. Montaner, et. Enhanced accumulation of anthocyanins in vitis vinifera cells immobilized in PUF. Enzyme Microb. Technol. 1994, 16(May): 416-419
[17] 李新明，侯崇生，陈士云等，九连小檗悬浮培养细胞的固定化研究，植物学报，1993，35(增刊)：101-105
[18] Taku Matsushita, Hiromichi Hidaka, Ken-ichi Kamihata, et. High density culture of anchorage-dependent animal cells by PUF packed-bed culture systems. Appl. Microbiol. Biotechnol.1991,35:159-164
[19] Robins RJ, Parr A J, Richards S R, Rhodes M J C (1986)Studies of environmental features of immobilized plant cell. In: Morris P, Scragg AH, Stafford A, Fowler M W (eds) Secondary Metabolism in Plant Cell Cultures, Cambridge University Press, Cambridge, PP162-172
[20] 武汉大学、复旦大学生物系微生物学教研室编，微生物学[第二版]，高等教育出版社
[21] 元英进，胡宗定：聚氨酯泡沫固定化长春花细胞生产生物碱的研究，化学反应工程与工艺，1994，10(2)，195-199
[22] 波钦诺克XH：植物生物化学分析方法，荆家海译，科学出版社，P145
[23] 中华人民共和国药典，中华人民共和国药典委员会，一九九五年版，一部，附录
[24] 郑玉果，采用愈伤组织固体培养法生产银杏内酯的初步研究，[学位论文]，1996
[25] Steinke, B. Müller, B. , Wager, H. , Planta Med. 1993, 59 (2) ,150 -160
[26]王佃亮，肖成祖，动物细胞高密度培养用多孔微球，生物工程进展，1998，18(2)，46-49．
[27] Messing, R.A,et al.Biotechnol. Bioeng., 1979,21,49-58