【作者】 孙祺；

【导师】 胡宗定； 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2004， 硕士

【摘要】 为了了解微囊固定化植物细胞的生长与代谢规律，为大规模固定化植物细胞生产奠定实验基础，本文以海藻酸-壳聚糖包埋东北红豆杉细胞（Taxuscuspidata），通过对照在不同胶囊包埋条件下与普通悬浮培养细胞的异同点，改变包埋量、包埋载体密度、换液时间等参数，观察了微囊化细胞生长与代谢情况，分析了微囊固定化细胞产生这些生长与代谢变化的主要影响因素，对胶囊包埋红豆杉细胞生长与代谢的影响形成一个整体的认识。得到如下结果： 用海藻酸-壳聚糖胶球包埋东北红豆杉细胞时，细胞受到最大影响在传质方面，细胞活力下降。海藻酸钠-壳聚糖液芯胶球和无壳聚糖层的海藻酸钠胶球作为改进包埋方法，有利于保持较高的细胞存活率和代谢能力，对糖类等营养物质的吸收较好。两者均使细胞产生一些应激反应，包括细胞膜通透性增大，胞外蛋白含量增加，培养液出现碱化等。而无论有无壳聚糖层包裹的固芯胶球均比液芯胶球更利于酚类等次生代谢产物的合成与积累。 细胞生长的停滞期换液不能明显改进细胞存活率以促进次生代谢产物的积累。换液后的细胞所出现的生理现象，包括细胞膜透性变化、蛋白质的吸收与代谢、酚积累等方面表现出与未换液相近似的规律。 选用不同海藻酸钠载体密度包埋细胞时，随着载体密度增大，培养液中的酚积累和电导率增长。20g/L-30g/L 是适中的载体密度，有利于保持包埋细胞较高的活力。细胞包埋的不同阶段，包埋量影响细胞存活率、营养消耗等的程度不同。高包埋量使细胞间相互刺激作用较大，有更多胞内离子向胞外释放。包埋量越高，其酚积累的峰值出现越晚。在细胞的停滞期，酚积累的量与包埋量正相关。 海藻酸钠胶球固定化细胞可以使其膜透性改变，胞内离子向外释放。这种刺激一方面是包埋在胶球表面的细胞受到其它胶球冲击、剪切等作用，另一方面是包埋在胶球内部的细胞受到包埋载体和其它细胞的相互作用。更多还原

【Abstract】 In order to make a deep understanding of growth and metabolism ofencapsulated cells, Taxus cuspidate cells were immobilized in variousalginate-chitosan beads. By alternating matrix density, inoculums, subculture, weanalysis reasons for particular cell physiology, and made a comprehensive study. Wegot results as following: Immobilization effected cells most on mass diffusion, and cells viability wasreduced. Liquefied beads and solid beads without chitosan, as improved immobilizedmeasure, maintained the viable cell ratio better, enhanced cells metabolism, andincreased absorption of sugar and other nutrition. They also stimulated immobilizedcells,so cells membrane penetration increased, extracellar protein improved and pH ofmedium fell. And solid beads without chitosan made the augment of cell penetrationless than liquefied beads. Solid beads with chitosan membrane or not strengthenedsecretion and accumulation of phenol more than liquefied beads. Subculture in cell lag stage in this experiment did not improve viable cells ratioto get more secondary metabolite production. Encapsulated cells in subcultureexhibited a few physiology behaviors same like original culture. Denser matrix stimulated encapsulated cells more; as a result phenolicsaccumulation was enhanced, and conductivity in medium increased. But densermatrix did harm to cells in long stage culture. So moderate matrix density(20g/L-30g/L) benefited cells to maintain high viability.Inoculums impacted viablecells ratio, nutrition consumption with different level in different period. In earlyculture time, the impaction was tiny. In lag stage, viable cells ration decreased wheninoculums increased. High inoculums stimulated cells more, and released moreintracellar ions. More inoculums delayed the peak of phenolics accumulation. Andphenolics accumulation had liner correlation with inoculums in lag stage. Alginate-chitosan immobilization enhanced medium conductivity, because thebeads impacted cell penetration and made more ions released out. This resulted bothfrom shearing and flushing to cells on the beads surface and impaction of cells ininner beads.更多还原