红豆杉细胞培养与紫杉醇生产

介绍了１９９１年红豆杉细胞培养第一个专利发表以来,特别是近两年,在红豆杉细胞培养研究方面所取得的巨大进展,其中特别强调提高紫杉醇含量方法的研究。目前,红豆杉细胞培养的规模和紫杉醇含量都已达到商业生产的水平,高技术产业的形成已为期不远。     

红豆杉细胞悬浮培养生产紫杉醇研究进展

介绍了近年来由红豆杉细胞培养生产紫杉醇领域取得的进展,其中特别介绍了由紫杉醇生物合成途径及其代谢酶类的研究发展而来的用基因工程方法改良细胞系,为提高紫杉醇产量而采取添加前体物质,诱导子,抑制子等方面的研究。     

南方红豆杉组织培养及紫杉醇的产生

南方红豆杉茎段愈伤组织在不同基本培养基及激素组合的培养基上,其平均鲜重生物量具显著差异,而干重生物量无差异。适宜浓度的ＮＡＡ可以取代常用的、但致癌活的２,４－Ｄ。     

东北红豆杉细胞培养生产紫杉醇的调控研究Ⅱ——交互作用对东北红豆杉细胞培养生产紫杉醇的影响分析

交互作用可能发生在两种相同或不同类型物质之间 ,其影响可能是促进也可能是滞后 ,研究交互作用可以更为深入地了解两种物质之间发生的具体影响 .考察了乙酸钠×丙酮酸钠、SA×CCC、乙酸钠×SA、乙酸钠×CCC、赤霉酸×CCC共 5种交互作用 ,较为全面地反映了典型交互作用中二物质之间的相互影响 .经进一步研究发现当两两物质之间达到某一定浓度的时候 ,必然会出现相互抑制的作用 .     

红豆杉组织培养及其产物紫杉醇研究进展

该文对红豆杉组织培养过程中外植体的选择、培养基、培养方法等方面研究进展进行了综述。获取抗癌药物紫杉醇主要途径有:红豆杉组织培养途径、人工种植途径、化学合成及微生物生产途径等。     

红豆杉紫杉醇提取方法的建立

目的：研究粉碎粒度、提取溶剂、提取时间、料液比等因素对红豆杉中紫杉醇得率的影响,建立一种高效、快速的提取和检测方法。方法：采用有机溶剂-超声波提取法从红豆杉叶片中提取紫杉醇,用HPLC法对其进行鉴定,用标准曲线法进行定量分析。结果：将原料于95％烘箱内干燥至恒重,粉碎至100目,以95％甲醇为提取剂,于60℃提取20h,料液比为1：10,紫杉醇得率为0．086mg／g。结论：该法简单、快速、准确、适应性强,为紫杉醇的提取与检测提供了有效工具。     

诱导子在红豆杉细胞培养生产紫杉醇中的应用研究进展

诱导子作为一种调控植物次生代谢产物生物合成的重要手段,用于红豆杉(Taxus)细胞培养生产紫杉醇的研究效果显著。本文分别就生物诱导子和非生物诱导子促进紫杉醇生物合成及其可能的作用机制进行了综述。     

红豆杉内生真菌产紫杉醇研究进展

本文综述了近年来产紫杉醇的红豆杉内生真菌分离和筛选、代谢和发酵的研究进展。     

红豆杉细胞两相培养生产紫杉醇的研究

研究了两相培养系统中红豆杉细胞的生长与代谢规律,经４０天培养,细胞生物量为１７．８５ｇ／Ｌ（干重）,紫杉醇产量为３０．１９ｍｇ／Ｌ。     

红豆杉细胞培养生产紫杉醇产量稳定性的探讨

通过磷酸盐双饥饿和秋水仙碱这两种经典的同步化方法处理悬浮培养的红豆杉细胞 ,以实现培养物的均一性 ,并比较了同步化与非同步化细胞及不同同步化方法处理的细胞紫杉醇产量。结果表明 ,不同同步化方法处理的细胞紫杉醇产量有差异 :秋水仙碱同步处理处于中期的细胞紫杉醇产量高于非同步化细胞 ,而磷酸盐双饥饿同步处理处于间期的细胞紫杉醇产量则相反。这表明紫杉醇产量与培养物的均一性有关 ,且与细胞同步的周期时相有关 ,采用同步化方法来选择合适的细胞周期时相有利于紫杉醇产量的稳定 ,通过比较不同同步化方法处理对细胞生物量和 POD活性的影响进一步探讨紫杉醇产量产生差异的原因     

红豆杉细胞培养中紫杉醇高产细胞株的筛选及其稳定性分析

对中国红豆杉〔Taxus chinensis(Pilger)Rehd.〕、云南红豆杉(T. yunnanensis Cheng et L.K.Fu)和东北红豆杉(T. cuspidata Sieb.et Zucc.)不同部位的愈伤组织进行培养及分析比较。结果表明,中国红豆杉愈伤组织生长较快,其叶片诱导的愈伤组织紫杉醇含量较高,且紫杉醇含量与培养物的外观特征有明显相关性,颜色浅、块状或颗粒较明显的细胞团紫杉醇含量较高。运用细胞看护培养技术,从中国红豆杉愈伤组织中筛选出生长速率达0.52g.L-1.d-1、紫杉醇含量超过0.01%的细胞优株,经20次继代培养,其生长和紫杉醇含量均较稳定。     

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.     

聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产的影响

在改良的B5培养基中加入不同浓度的聚乙二醇对东北红豆杉培养细胞进行摇瓶培养,通过不同时期取样并测定细胞鲜,干重及用HPLC测定紫杉醇的含量,发现聚乙二醇对东北红豆杉培养细胞的生长及紫杉醇生产均有明显的促进作用,聚乙二醇为10g/L时,对细胞生长最为有利,细胞培养16d可达到最大生物量,其平均鲜重为28．73g/瓶,增重3．8倍,平均干重为2．14g/瓶,增重3．1倍,聚乙二醇为20g/L,对紫杉醇的生产最有利;细胞培养25d时,培养基中紫杉醇的含量达到最高水平,其含量为2350ug/L,是不加聚乙二醇的11倍。     

Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.     

云南红豆杉(Taxus Yunnanensis)内生真菌中产紫杉醇真菌的筛选

报道了从云南红豆杉（Taxus Yunnanensis)树皮和枝条中分离得到的52株内生真菌中,有19株菌的发酵产物经TLC分析和HPLC检测验证后产紫杉醇或紫杉烷类物质。其中紫杉醇含量在1‰以上的菌株有8株。TAX-47（茎点霉属）和TAX-49（组丝核菌属）的紫杉醇得率分别为47.302μg/L和31.06μg/L,TAX-25（茎叶核菌属）巴可亭Ⅲ得率为744.2μg/L,这些菌株值得进一步研究。     

稀土元素对红豆杉细胞悬浮培养及紫杉醇合成的影响

研究了在250mL摇瓶中,不同浓度的硝酸镧、硫酸铈铵、硝酸亚铈3种稀土化合物对细胞生长及紫杉醇分泌和释放的影响。结果表明,在培养初期加入稀土元素。3种不同稀土化合物对细胞生长影响强弱不同,但趋势相似,均使细胞的延迟期缩短。1ppm的Ce^4 促进细胞生长的效果最明显。细胞干重第17d达到10.9g/L。在指数期加入稀土元素。10ppmCe^3 刺激细胞生长的效果最明显,细胞干重最高值达到11.5g/dL,比对照高1.5g/L,而10ppm的La^3 抑制细胞的生长。经稀土元素处理后,细胞胞内和胞外紫杉醇含量都有大幅度的提高,其中以10ppmCe^3 处理,胞外紫杉醇释放率最大,达37.7%。     

云南红豆杉中紫杉醇和四种紫杉烷类化合物含量

云南红豆杉中紫杉醇和四种紫杉烷类化合物含量项伟张宏杰阮德春孙汉董（云南省林业科学院,昆明６５０２０４）（中国科学院昆明植物研究所,昆明６５０２０４）Ｃｏｎｔｅｎｔｓｏｆｔａｘｏｌａｎｄｏｔｈｅｒ４ｔａｘａｎｅｄｉｔｅｒｐｅｎｏｉｄｓｉｎＴａｘｕｓｙｕ...     

云南省红豆杉资源的分布,利用现状与保护和可持续利用

红豆杉属（ＴａｘｕｓＬ．）植物是提取抗癌新药紫杉醇（ｔａｘｏｌ）的原料,在云南省有２种１变种,分布范围较广,但多散生,野生数量计有３５４０９２０株。近年来,红豆杉资源的开发利用研究进展较快,但资源的破坏和浪费严重,加之该植物生长缓慢,对生境的要求高,营造原料林困难,供求矛盾十分突出,为此提出了红豆杉资源的保护和可持续利用对策。     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011