云南红豆杉细胞的悬浮培养

在云南红豆杉细胞悬浮培养中,适宜的培养基为B5,接种量为0．5～0．8g干重细胞／100ml培养基,2,4－D浓度为1．0mg／L;培养细胞的生长周期约30d;培养基中较高浓度的蔗糖（40g／L）可提高紫杉醇含量;添加的椰子汁（CM）、酪蛋白氨基酸（C）和水解乳蛋白（LH）3种有机添加剂均能提高培养细胞中紫杉醇的含量,但只有CM和CA能促进细胞的生长。于B5培养基中添加不同浓度的NH4NO3对培养细胞无明显影响。     

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。     

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%。     

红豆杉悬浮细胞放大培养的细胞生长与紫杉醇合成动力学

研究了在Murashige&skoog s(MS)和 6 2号两种不同的培养基中 ,红豆杉细胞悬浮细胞从摇瓶到 1 0L机械通气搅拌式反应器放大培养过程中细胞生长与紫杉醇合成动力学 .结果表明 :尽管在不同的培养条件下 ,细胞生长曲线均呈现“S”型 .紫杉醇在延迟期与指数生长期中基本上没有积累 ,而且随着培养规模的增大 ,紫杉醇的含量逐渐降低 .进一步对各级放大培养的细胞生长 ,比生长率与胞内外紫杉醇合成量进行分析 ,发现MS利于细胞生长但不利于紫杉醇合成 ,而 6 2号则相反 .根据此文的结果 ,提出了红豆杉细胞培养条件的优化和大规模细胞培养生产紫杉醇应采取的策略     

红豆杉细胞悬浮培养中不同添加物对细胞生长和紫杉醇合成的影响

采用正交实验检测红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ）Ｒｅｈｄ．）细胞悬浮培养中水杨酸、Ｄ－果糖、甘露醇和硫酸镧对细胞生长和紫杉醇（ｔａｘｏｌ）积累的影响。添加１０ｇ／ＬＤ－果糖,可使细胞的鲜重和干重明显增加;添加６０ｇ／Ｌ甘露醇使细胞的鲜重和干重明显减少;１ｍｇ／Ｌ水杨酸仅使细胞鲜重增加,对干重影响不明显;硫酸镧对细胞生长无明显影响。单独添加这４种物质,紫杉醇含量均下降,同时添加     

云南红豆杉细胞的悬浮培养

在云南经豆杉细胞悬浮培养中,适宜的增减基为Ｂ５,接种量为０．５－０．８ｇ干重细胞／１００ｍｌ培养基,２,４－Ｄ浓度为１．０ｍｇ／Ｌ;培养细胞的生长周期约３０ｄ;增减基中较高浓度的蔗糖（４０ｇ／Ｌ）可提高紫杉醇含量;添加的椰子汁（ＣＭ）、酪蛋白氨基酸（ＣＡ）和水解乳蛋白（ＬＨ）３种有机添加剂均能提高培养细胞中紫杉醇的含量,但只有ＣＭ和ＣＡ能促进细胞的生长。于Ｂ５培养基中添国不同浓度的ＮＨ４ＮＯ３对培     

DMSO对悬浮培养的东北红豆杉细胞凋亡和紫杉醇合成的影响

研究了不同浓度的DMSO对悬浮培养的东北红豆杉（Taxus cuspidata）细胞的增殖能力、细胞活性以及紫杉醇合成和释放等方面的影响,同时应用荧光指示剂双染法检测了细胞凋亡的发生情况。结果显示2％的DMSO处理能显著降低细胞活性,抑制细胞的增殖能力,使细胞核内DNA含量减少,培养中、后期在荧光显微镜下可见部分细胞核出现典型的凋亡形态,同时伴有紫杉醇产量的明显增加。对照组及1％以下浓度组未出现上述改变。结果表明一定浓度的DMSO能诱导细胞凋亡,促进细胞紫杉醇合成能力的提高。     

利用运动红豆杉悬浮细胞生长紫杉醇和紫杉烷类化合物

云南红豆杉（Taxus yunnanensis Cheng et L.K.Fu）的一些株紫杉醇高产细胞系经过8年多的继代培养,仍保持较稳定的紫杉烷类化合物的生物合成能力。从此株紫杉醇高产细胞系的悬浮培养物中分离到8个紫杉烷类合物。经核磁共振光谱和质谱数据分析,它们的化学结构分别是2,5,10－三乙酰基－14－丙酰氧基紫杉二烯（1）、2,5,10－三酰氧基－14－（2′－甲基丙酰氧基）紫杉二烯（2）,2,5,10,14－四乙酰氧基紫杉二烯（3）、2,5,10－三乙酰氧基－14－（2′－甲基－3′－羟基丁酰氧基）紫杉二烯及其差向异构体（4和5）、巴卡亭Ⅳ（6）、巴卡亭Ⅲ（7）和紫杉醇（8）。化合物3、5－7为首次从云南红豆杉细胞培养物中分离到。定性分析表明,云南红豆杉细胞悬浮培养液中的化学成分与培养细胞中的相似。另外,此株紫杉高产细胞系的紫杉醇含量可达高0．3％,可用来进行大规模培养。     

南方红豆杉细胞悬浮培养条件优化研究

针对红豆杉细胞生长相对缓慢和培养褐化问题,本文通过正交实验和均匀实验方法并采用人工神经网络技术对南方红豆杉细胞悬浮培养条件进行优化,使细胞的生长速率和比生长速率有较大提高,并考察了细胞活性随时间的动态变化.     

中国红豆杉悬浮培养细胞聚集体异质性的初步研究

通过梯度浓度的蔗糖溶液（0－1mol/L）的筛选,分拣出7种不同物理学密度的红豆杉细胞聚集体,并对它们进行了木质素含量及紫杉醇含量测定。结果显示：不同的细胞聚集体在物理学密度、木质素含量、紫杉醇含量、生长速度等方面存在着差异,其中密度最小的细胞聚集体木质素及紫杉醇含量分别是密度最大的细胞聚集体的5倍和8倍,并且在一定密度范围内（大于0.2mol／L蔗糖溶液密度）,细胞聚集体的木质素含量与紫杉醇的含量呈平行关系,表明不同聚集体紫杉醇含量一细胞分化有一定的关系,首次提出了红豆杉悬浮细胞聚集体培养中存在着异质现象,并对其可能机理及意义给予阐述。     

中国红豆杉悬浮培养细胞的超低温保存

对中国红豆杉悬浮细胞超低温保存中几个主要因素进行多方面对比研究。结果表明,取培养16d的细胞进行超低温保存效果最好,10％DMSO＋8％葡萄糖作为冰冻保护剂对冷冻细胞起到最佳的保护效果;较好的降温程序是在0℃中预处理30min后移入－20℃中停留180min,然后转入－70℃中停留30min,最后投入－196℃液氮中保存。该实验还对保存后细胞的恢复性生长进行了验证。     

A structured kinetic model for suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum

A structured kinetic model was established to describe the process of Taxol formation in suspension cultures of Taxus chinesis var. mairei induced by an oligosaccharide from Fusarium oxysporum. In this model, the role of intracellular starch as a storage carbon source had to be taken into account. Substrate uptake, culture growth, cell respiration, and secondary metabolites, predicted by the model, agreed with those obtained experimentally. The effective factors of oligosaccharide elicitation, _e,j, defined as the ratio of the parameter values in the system with oligosaccharide to those in control, reflected the effects of the oligosaccharide on cell growth and Taxol production.     

石竹细胞悬浮培养研究

石竹细胞继代周期为 7d时 ,悬浮细胞培养系生长最快 ,生长率最高 ,而且培养物中胚性细胞较多 ,并能保持较快的分裂和生长 ,能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以1∶2较好 ,悬浮系细胞生长最快 ,生长率最高 ,以 1∶2和 1∶3的高倍稀释接种有利于胚性细胞的形成及产生小的胚性细胞团 ,对悬浮系添加椰乳和水解乳蛋白的混合物 ,可较大幅度地提高悬浮细胞系的生长速率 ,单独添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中 ,配方 2 (2 ,4 D 1 .5mg/L +NAA0 .5mg/L +6 BA 0 .5mg/L)最好 ,生长率最高。配方 5 (2 ,4 D 1 .5mg/L +NAA 0 .5mg/L +6 BA 1 .0mg/L)其次 ;配方 1 (2 ,4 D 1 .0mg/L +NAA 0 .5mg/L +6 BA 0 .5mg/L)次之。     

原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产

本实验所用的中国红豆杉细胞悬浮培养体系中,云南紫杉烷c(Tc)是主要的次生代谢产物,该化合物有类神经生长因子活性,提高其产量是进一步规模化生产的前提。本研究考察了原位吸附和茉莉酸甲酯(MJA)联合调控提高Tc产量的可能性。在培养的第7天加入浓度为100μmol/L的MJA虽然会使细胞的生物量下降10%～30%,但是单位细胞内Tc含量和Tc产量均有显著提高,分别是对照的3.6和3.3倍。吸附剂XAD-7在不同时间加入对Tc的合成影响显著。在培养的第7天同时加入100μmol/L的MJA和100g/L的XAD-7会使细胞生物量增加,Tc产量显著提高。培养到第21天,Tc产量达477.4mg/L,为对照的6.3倍,为只加MJA的1.9倍,其中94%的Tc被树脂吸附。实验结果表明,在MJA诱导高表达的过程中,吸附剂XAD-7的加入使细胞内代谢产物外泌,浓度降低,减轻产物反馈抑制现象,从而大幅度提高代谢物产量,有较好的生产前景。     

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.     

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     

Salicylic acid-induced taxol production and isopentenyl pyrophosphate biosynthesis in suspension cultures of Taxus chinensis var. mairei

The influences of salicylic acid (SA) on taxol production and isopentenyl pyrophosphate (IPP) biosynthesis pathways in suspension cultures of Taxus chinensis var. mairei were investigated by adding SA and mevastatin (MVS), a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase in the mevalonate pathway for IPP biosynthesis, into the culture systems. The cell death and taxol production were induced upon the introduction of SA, and 20mg/l was proved to be the optimal SA concentration in terms of the less damage to Taxus cells and marked activation of phenylalanine ammonia lyase (PAL). In the coexistence of SA (20mg/l) and MVS (100 nmol/l), the taxol content (1.626 mg/g dry wt) was higher than that (0.252 mg/g dry wt) of the MVS-treated system but almost equal to that (1.581 mg/g dry wt) of the SA-treated system. It is thus inferred that the activated non-mevalonate pathway should be responsible for the formation of IPP in taxol biosynthesis in the presence of SA.     

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

Application of a new structured model to tobacco cell cultures

A new structured kinetic model has been formulated and applied to batch suspensions of Nicotiana tabacum. This model has been developed by representing culture interactions with pathways designated for structural component production, secondary metabolite synthesis, and cellular respiration. Additional provisions were made to distinguish growth-competitive secondary metabolite production from non-growth-competitive secondary metabolite production. Parameters for kinetic rate expressions within the model were estimated based upon experimental observations utilized in conjunction with numerical optimization techniques. Using these parameters, culture growth, substrate uptake, cell respiration, and total phenolics production were all successfully correlated to experimenta data from shake flask cultures of N. tabacum.