红豆杉细胞培养中紫杉醇代谢调节的研究
摘要
紫杉醇是从红豆杉植物中提取的一种新型抗癌药物。紫杉醇在植物组织中的含量很低，而且植物组织或其细胞培养物的粗提样品中含有较多的杂质不宜分离。高效液相色谱(HPLC)逐渐显示出在分离、检测紫杉醇和BacctinⅢ等化合物方面的优势。采用岛津SPD-10AVP型HPLC，乙腈：水梯度洗脱检测，可以精确分离紫杉醇及其前体物质，紫杉醇和BacctinⅢ在0.01-0.25mg／ml范围内峰面积和浓度线性良好，结果标准曲线方程为：紫杉醇C=2.4×10~(-8)×S-0.002；BaccatinⅢC=2.9×10~(-8)×S-0.014。精密度RSD＜2％(n=5)。
植物生长调节物质在细胞生长和物质代谢过程中起着重要的作用。高浓度的NAA不利于紫杉醇的合成，而降低NAA的浓度对愈伤组织的生长影响较小。当Z_3激素浓度NAA：6-BA为2：0.5时，紫杉醇含量明显增高，而生长率仅稍有降低。因此，选用NAA 2.0mg／l，6-BA 0.5mg／l作为Z_3细胞生产紫杉醇的激素组合。液体培养条件下，接种量是影响细胞生长和代谢的重要因子。高的细胞接种量(8g／40ml)虽然有较低的平均生长率，但是它有较高的生物量合成，紫杉醇的含量也很高。
实验证实在液体培养时BacctinⅢ大部分外渗。细胞BacctinⅢ外渗与细胞状态并无相关性。液体培养的细胞整个生长周期内细胞活力比较稳定，在生长周期的第12天时即对数生长期末，细胞活力开始下降，此时可以作为细胞继代的最佳时期。
培养基的pH值影响细胞的生长、代谢。液体培养时细胞生长迅速，pH值先下降后上升。实验表明灭菌前培养基pH值为6.0，是细胞生长、代谢的最适pH值。培养基中加入pH值缓冲液，可以使pH相对稳定，改善细胞BacctinⅢ外渗，提高细胞中的紫杉醇含量。在此基础上添加紫杉
河北师范大学2003届硕士研究生学位论文
醇侧链前体，能进一步促进紫杉醇的合成。此外降低培养基渗透压的方法
亦能改善细胞Bacctinln外渗，使紫杉醇有所提高。
电镜观察结果表明植物细胞的超微结构和细胞代谢特征、次生代谢物
的积累有明显相关。次生代谢物含量高的细胞中有类似的结构特征:细胞
质浓厚，内容丰富，细胞器丰富，质体发达;次生代谢物含量低的细胞则
相反。采用合适材料，可以进一步揭示植物培养物分化与次生代谢产物积
累的相关性。
Taxol is a new type of anti-cancer medicine. But the concentration of taxol in taxus plants is relatively low. Furthermore, there are many by-productions in distilled substance of plant or cultivated cells. HPLC has advantages on testing these compounds. It indicated that the relationship between the peak area and the concentration was linearity. The criterion cure of taxol was C = 2. 4 10-8 S-0. 002 BaccatinⅢ C=2.9 10-8 S-0. 014 .
Growth hormones have an important effect on growth and metabolize of cell. High concentration of NAA was disadvantageous to the form of taxol. It had few effects on the growth rate when its concentration was reduced. The content of taxol was increase remarkably when NAA:6~BA is 2:0.5 at the same time there was only a little decline on growth rate. Higher biomass accumulation and taxol yield were observed in higher inoculate volume though the growth rate was lower.
The cell activity was stable in growth cycle and independent to Bacctinlll exosmosis. It is the appropriate time to subculture when the cell activity begin to drop. pH has effects on cell growth and metablism. During the growth of cells, the pH of medium decreased in the begining and increased then.
We can reduce the Bacctin Ⅲ exosmosis and increase taxol accumulation by such methods as follows: add MES to medium, reduce osmotic pressure and add precursor of said chain.
The ultrastructure and accumulation of taxoids were correlative.
6 -
The ultrastructures in cells of high level of metablism were same: cytoplasm was strong and abundant, plastids were numerous. The relationship of accumulation of productions of secondary metablism and the differentiation of plant tissue will be clear if appropriate materials were used.
植物生长调节物质在细胞生长和物质代谢过程中起着重要的作用。高浓度的NAA不利于紫杉醇的合成，而降低NAA的浓度对愈伤组织的生长影响较小。当Z_3激素浓度NAA：6-BA为2：0.5时，紫杉醇含量明显增高，而生长率仅稍有降低。因此，选用NAA 2.0mg／l，6-BA 0.5mg／l作为Z_3细胞生产紫杉醇的激素组合。液体培养条件下，接种量是影响细胞生长和代谢的重要因子。高的细胞接种量(8g／40ml)虽然有较低的平均生长率，但是它有较高的生物量合成，紫杉醇的含量也很高。
实验证实在液体培养时BacctinⅢ大部分外渗。细胞BacctinⅢ外渗与细胞状态并无相关性。液体培养的细胞整个生长周期内细胞活力比较稳定，在生长周期的第12天时即对数生长期末，细胞活力开始下降，此时可以作为细胞继代的最佳时期。
培养基的pH值影响细胞的生长、代谢。液体培养时细胞生长迅速，pH值先下降后上升。实验表明灭菌前培养基pH值为6.0，是细胞生长、代谢的最适pH值。培养基中加入pH值缓冲液，可以使pH相对稳定，改善细胞BacctinⅢ外渗，提高细胞中的紫杉醇含量。在此基础上添加紫杉
河北师范大学2003届硕士研究生学位论文
醇侧链前体，能进一步促进紫杉醇的合成。此外降低培养基渗透压的方法
亦能改善细胞Bacctinln外渗，使紫杉醇有所提高。
电镜观察结果表明植物细胞的超微结构和细胞代谢特征、次生代谢物
的积累有明显相关。次生代谢物含量高的细胞中有类似的结构特征:细胞
质浓厚，内容丰富，细胞器丰富，质体发达;次生代谢物含量低的细胞则
相反。采用合适材料，可以进一步揭示植物培养物分化与次生代谢产物积
累的相关性。
Taxol is a new type of anti-cancer medicine. But the concentration of taxol in taxus plants is relatively low. Furthermore, there are many by-productions in distilled substance of plant or cultivated cells. HPLC has advantages on testing these compounds. It indicated that the relationship between the peak area and the concentration was linearity. The criterion cure of taxol was C = 2. 4 10-8 S-0. 002 BaccatinⅢ C=2.9 10-8 S-0. 014 .
Growth hormones have an important effect on growth and metabolize of cell. High concentration of NAA was disadvantageous to the form of taxol. It had few effects on the growth rate when its concentration was reduced. The content of taxol was increase remarkably when NAA:6~BA is 2:0.5 at the same time there was only a little decline on growth rate. Higher biomass accumulation and taxol yield were observed in higher inoculate volume though the growth rate was lower.
The cell activity was stable in growth cycle and independent to Bacctinlll exosmosis. It is the appropriate time to subculture when the cell activity begin to drop. pH has effects on cell growth and metablism. During the growth of cells, the pH of medium decreased in the begining and increased then.
We can reduce the Bacctin Ⅲ exosmosis and increase taxol accumulation by such methods as follows: add MES to medium, reduce osmotic pressure and add precursor of said chain.
The ultrastructure and accumulation of taxoids were correlative.
6 -
The ultrastructures in cells of high level of metablism were same: cytoplasm was strong and abundant, plastids were numerous. The relationship of accumulation of productions of secondary metablism and the differentiation of plant tissue will be clear if appropriate materials were used.
引文
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2．郭志刚，冯莹，刘瑞芝．生长调节物质对紫杉醇和紫杉烷类化合物合成的调控作用，天然产物开发与研究，2000，12(3)：22—26
3．韩金玉，肖剑，王华．紫杉醇的提取及纯化技术，中草药，2000，31(11)
4．侯学文，郭勇．接种量及蔗糖浓度对悬浮培养玫瑰茄细胞生长的影响，华南农业大学学报，2000，21(4)
5．胡国武，元英进．硝酸铈对红豆杉细胞培养及紫杉醇合成的影响，稀土，2000，21(2)：49—51
6．胡国武，元英进．紫杉醇合成代谢途径中紫杉烯合成酶cDNA的克隆，生物工程学报，2002
7．黄巧明．改进的H P L c法测定红豆杉植物和细胞培养物中紫杉醇的含量，植物资源与环境学报，2002，11(1)：53-54
8．柯铁，梅兴国．HPLC法检测红豆杉细胞培养物中的紫杉醇，生物技术通报，2000，5：26-29
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