喜树细胞培养体系的建立及培养动力学

【作者】 于放；

【导师】 白凤武；

【作者基本信息】 大连理工大学 ， 生物工程， 2001， 硕士

【摘要】 本论文针对喜树，以具有特效抗癌活性的喜树碱及其它可能具有生物学活性的次级代谢产物为目标产物，研究了喜树细胞愈伤组织诱导、生物反应器培养、次级代谢产物分离纯化等诸多内容，包括： 以喜树茎、叶为外植体共诱导出多种愈伤组织。通过改变激素配比等策略，对喜树愈伤组织继代培养，成功地获得了适合悬浮培养的松散型喜树愈伤组织颗粒。 建立了喜树细胞悬浮培养系，考察其特性发现：细胞自凝聚形成的颗粒尺寸主要集中在2—2.5mm之间且符合正态分布，适宜于生物反应器中进行高密度培养，气升式生物反应器中的氧传递速率能够满足喜树细胞颗粒生长的需求。 系统研究了喜树细胞颗粒在生物反应器中培养的动力学规律，建立了喜树细胞生长的结构化动力学模型。模型由颗粒结构组织、颗粒内游离磷酸盐、积累的淀粉、分生点及代谢产物等5个部分组成。模型中喜树细胞的生长受培养基C源及颗粒内磷酸盐两种底物浓度的限制；代谢产物合成的速率则主要与分生细胞的生长速率有关；颗粒内磷酸盐浓度在喜树细胞的生长、颗粒内淀粉的积累以及代谢产物生物合成等过程中起调节作用。 对喜树细胞自凝聚颗粒培养过程次级代谢产物的分析发现，原植株中的喜树碱在细胞培养过程中没有积累，但是培养物中呈现二种原植株中没有的新物质，初步检测具有生物学活性。我们对这二种新物质进行了分离纯化，通过硅胶柱的粗分离和制备色谱的精分离，得到了纯品，有待于进一步的结构分析。更多还原

【Abstract】 Six different kinds of Camptotheca acuminata calli were successfully initiated from the explants based on the stems and leaves. A callus line with a size of 2?.5 mm in diameter and suitable for suspension culture in the bioreactors was developed by manipulating of the ratio of the plant regulators. Then, the Camptotheca acuminata callus suspension cultures were carried out using an air-lift bioreactor with a working volume of 5L in order to produce Camptothecine, a anti- cancer drug. The results showed that oxygen mass transfer performance of the bioreactor is good enough for the callus particle growth at high density (12 g (d.w) /L). Furthermore, structured kinetics of Camptotheca acuminafa callus growth and secondary rnetabolite formation were proposed based on the analysis of metabolism pathways. Finally, the separation and purification processes of secondary metabolites were developed based on thinning silicon gel chromatography and HPLC. Although predicted aimed梡roduct, Camptothecine, cannot be detected, two new secondary metabolites which have been tested to have bioactivity (anti-cancer) were successfully discovered within the harvested cultures.更多还原