1、 Characterization of production of Paclitaxel and related Taxanes in Taxus Cuspidata Densiformis suspension cultures by LC, LC/MS, and LC/MS/MS CHAPTER THEREPLANT TISSUE CULTURE. Potential of Plant cell Culture for Taxane ProductionSeveral alternative sources of paclitaxel have been identified and a
2、re currently the subjects of considerable investigation worldwide. These include the total synthesis and biosynthesis of paclitaxel, the agriculture supply of taxoids from needles of Taxus species, hemisynthesis (the attachment of a side chain to biogenetic precursors of paclitaxel such as baccatin
3、or 10-deacetylbaccatin ), fungus production, and the production of taxoids by cell and tissue culture. This reciew will concentrate only on the latter possibility.Plant tissue culture is one approach under investigation to provide large amounts and a stable supply of this compound exhibiting antineo
4、plastic activity. A process to produce paclitaxel or paclitaxel-like compounds in cell culture has already been parented. The development of fast growing cell lines capable of producing paclitaxel would not only solve the limitations in paclitaxel supplies presently needed for clinical use, but woul
5、d also help conserve the large number of trees that need to be harvested in order to isolate it. Currently, scientists and researchers have been successful in initiating fast plant growth but with limited paclitaxel production or vice versa. Therefore, it is the objective of researchers to find a me
6、thod that will promote fast plant growth and also produce a large amount of paclitaxel at the same time. Factors Influencing Growth Paclitaxel Content A. Choice of Media for Growth Gamborgs (B5) and Murashige & Skoogs (MS) media seem to be superior for callus growth compared to Whites (WP) medium. T
7、he major difference between these two media is that the MS medium contains 40 mM nitrate and 20mM ammonium, compared to 25mM nitrate and 2mM ammonium. Many researchers have selected the B5 medium over the MS medium for all subsequent studies, although they achieve similar results. Gamborgs B5 media
8、was used throughout our experiments for initiation of callus cultures and suspension cultures due to successful published results. It was supplemented with 2% sucrose, 2 g/L casein hydrolysate, 2.4 mg/L picloram, and 1.8 mg/L -naphthalene acetic acid. Agar (8 g/L) was used for solid cultures.B. Init
9、iation of Callus Cultures Previous work indicated that bark explants seem to be the most useful for establishing callus. The age of the tree did not appear to affect the ability to initiate callus when comparing both young and old tree materials grown on Gamborgs B5 medium supplemented with 1-2 mg/L
10、 of 2,4-dichlorophenoxyacetic acid. Callus cultures initiated and maintained in total darkness were generally pale-yellow to light brown in color. This resulted in sufficient masses of friable callus necessary for subculture within 3-4 weeks. However, the growth rate can decline substantially follow
11、ing the initial subculture and result in very slow-growing, brown-colored clumps of callus. It has been presumed that these brown-colored exudates are phenolic in nature and can eventually lead to cell death. This common phenomenon is totally random and unpredictable. Once this phenomenon has been t
12、riggered, the cells could not be saved by placing them in fresh media. However, adding polyvinylpyrrolidone to the culture media can help keep the cells alive and growing. Our experience with callus initiation was similar to those studies.Our studies have found that callus which initiated early (usu
13、ally within 2 weeks ) frequently did not proliferate when subcultured and turned brown and necrotic. In contrast, calli which developed from 4 weeks to 4 months after explants were fist placed on initiation media were able to be continuously subcultured when transferred at 1-2 month intervals. The p
14、resence of the survival of callus after subsequent subculturing. The relationship between paclitaxel concentration and callus initiation, however, has not been clarified.C. Effect of Sugar Sucrose is the preferred carbon source for growth in plant cell cultures, although the presence of more rapidly
15、 metabolized sugar such as glucose favors fast growth. Other sugars such as lactose, galactose, glucose, and fructose also support cell growth to some extent. On the other hand, sugar alcohols such as mannitol and sorbital which are generally used to raise the sugars added play a major role in the p
16、roduction of paclitaxel. In general, raising the initial sugar levels lead to an increase of secondary metabolite production. High initial levels of sugar increase the osmotic potential, although the role of osmotic pressure on the synthesis of secondary metabolites is not cleat. Kim and colleagues
17、have shown that the highest level of paclitaxel was obtained with fructosel. The optimum concentration of each sugar for paclitaxel production was found to be the same at 6% in all cases. Wickremesinhe and Arteca have provided additional support that fructose is the most effective for paclitaxel pro
18、duction. However, other combinations of sugars such as sucrose combined with glucose also increased paclitaxel production.The presence of extracellular invertase activity and rapid extracellular sucrose hydrolysis has been observed in many cell cultures. These reports suggest that cells secrete or p
19、ossess on their surface excess amounts of invertase, which result in the hydrolysis of sucrose at a much faster rate. The hydrolysis of sucrose coupled with the rapid utilization of fructose in the medium during the latter period of cell growth. This period of increased fructose availability coincid
20、ed with the faster growth phase of the cells.D. Effect of Picloram and Methyl JasmonatePicloram (4-amino-3.5.6-trichloropicolinic acid) increases growth rate while methyl jasmonate has been reported to be an effective elicitor in the production of paclitaxel and other taxanes. However, little is kno
21、wn about the mechanisms or pathways that stimulate these secondary metabolites.Picloram had been used by Furmanowa and co-workers and Ketchum and Gibson but no details on the effect of picloram on growth rates were given. Furmanowa and hid colleagues observed growth of callus both in the presence an
22、d absence of light. The callus grew best in the dark showing a 9.3 fold increase, whereas there was only a 2-4 fold increase in the presence of light. Without picloram, callus growth was 0.9 fold. Unfortunately,this auxin had no effect on taxane production and the high callus growth rate was very un
23、stable.Jasmonates exhibit various morphological and physiological activities when applied exogenously to plants. They induce transcriptional activation of genes involved in the formation of secondary metabolites. Methyl jasmonate was shown to stimulate paclitaxel and cephalomannine (taxane derivativ
24、e) production in callus and suspension cultures. However, taxane production was best with Whites medium compared to Gamborgs B5 medium. This may be due to the reduced concentration of potassium nitrate and a lack of ammonium sulfate with Whites medium.E. Effect of Copper Sulfate and Mercuric Chlorid
25、eMetal ions have shown to play significant roles in altering the expression of secondary metabolic pathways in plant cell culture. Secondary metabolites,such as furano-terpenes, have been production by treatment of sweet potato root tissue with mercuric chloride. The results for copper sulfate, howe
26、ver, have not been reported.F. Growth Kinetics and Paclitaxel Production Low yields of paclitaxel may be attributed to the kinetics of taxane production that is not fully understood. Many reports stated inconclusive results on the kinetics of taxane production. More studies are needed in order to qu
27、antitate the taxane production. According to Nett-Fetto, the maximum instantaneous rate of paclitaxel production occurred at the third week upon further incubation. The paclitaxel level either declined or was not expected to increase upon further incubation. Paclitaxel production was very sensitive
28、to slight variations in culture conditions. Due to this sensitivity, cell maintenance conditions, especially initial cell density, length of subculture interval, and temperature must be maintained as possible.Recently, Byun and co-workers have made a very detailed study on the kinetics of cell growt
29、h and taxane production. In their investigation, it was observed that the highest cell weight occurred at day 7 after inoculation. Similarly, the maximum concentration for 10-deacetyl baccatin and baccatin were detected at days 5 and 7, respectively. This result indicated that they are metabolic int
30、ermediates of paclitaxel. However, paclitaxels maximum concentration was detected at day 22 but gradually declined. Byun and his colleagues suggested that paxlitaxel could be a metabolic intermediate like 10-deacetyl baccatin and baccatin or that pacliltaxel could be decomposed due to cellular morph
31、ological changes or DNA degradation characteristic of cell death.Pedtchankers group also studied the kinetics of paclitaxel production by comparing the suspension cultures in shake flasks and Wilson-type reactors where bubbled air provided agitation and mixing. It was concluded that these cultures o
32、f Taxus cuspidata produced high levels of paclitaxel within three weeks (1.1 mg/L per day ). It was also determined that both cultures of the shake flask and Wilson-type reactor produced similar paclitaxel content. However, the Wilson-type reactor had a more rapid uptake of the nutrients (i.e. sugar
33、s, phosphate, calcium, and nitrate). This was probably due to the presence of the growth ring in the Wilson reactor. Therefor, the growth rate for the cultures from the Wilson reactor was only 135 mg./L while the shake flasks grew to 310 mg/L in three weeks.In retrospect, strictly controlled culture
34、 conditions are essential to consistent production and yield. Slight alterations in media formulations can have significant effects upon the physiology of cells, thereby affecting growth and product formation. All of the manipulations that affect growth and production of plant cells must be carefull
35、y integrated and controlled in order to maintain cell viability and stability.利用 LC,LC/MS 和 LC/MS/MS 悬浮培养生产紫杉醇及邓西佛米斯红豆杉中相关紫杉醇类的特征描述第三章植物组织培养 .利用植物细胞培养生产紫杉的可能性紫杉醇的几个备选的来源已被确定，而且目前是全球大量调查的主题。这些来源包括人工合成紫杉醇，农业提供针头红豆杉紫杉，半合成体（紫杉醇生物前体的一条侧链的附属物，例如浆果赤霉素 或 10-去乙酰浆果赤霉素 ），真菌生产，以及通过细胞和组织培养生产紫杉。 这次审查将只集中在后者的可能性上。
36、植物组织培养是调查中用以提供大量稳定数量的这种具有抗肿瘤活性化合物的一种方法。用细胞培养生产紫杉醇或者紫杉醇样化合物的方法已获得专利。能够生产紫杉醇的的快速增长的细胞系的发展不仅能解决现在在临床应用上紫杉醇供应受限的问题，而且将有助于保护大量为了使之隔离需要收获的树。然而，植物组织培养技术生产紫杉醇的速度不能满足需求。目前，科学家和研究员已成功地启用了植物快速生长的方法，但这种方法紫杉醇的生产有限，反之亦然。因此，研究者的目标是找到一种能促进植物快速生长同时也能生产大量紫杉醇的方法。影响植物生长和紫杉醇含量的因素A 生长培养基的选择B5 和 MS 培养基与 WP 培养基相比似乎更有利于愈伤组织
37、生长。这两种培养基的主要区别在于 MS 培养基包含 40mM 硝酸盐 和 20mM 铵， B5 培养基中包含 25mM 硝酸 盐和 2mM 铵。相比之下，WP 培养基包含 10mM 硝酸盐和 5mM铵.一些研究者在所有随后的研究中选择 B5 培养基的数量超过 MS 培养基，尽管他们获得的结果相似。由于成功的公布的结果，B5 培养基在开始愈伤组织 培养和悬浮培养的整个实验中使用。它增补了 2的蔗糖，2g/L 干酪素水解物，2.4mg/L 毒莠定和1.8mg/L 萘乙酸。琼脂（8g/L ）用于固体培养。B. 愈伤组织培养的开始以往的工作表明树皮外植体似乎是对建立愈伤组织最有用的。当我们比较生长在补
38、充了 1-2mg/L 2，4-二氯苯氧乙酸的 B5 培养基中年轻的和老的树材料时，我们发现树龄并不影响 创造愈伤组织的能力。完全在黑暗中培养的愈伤组织一般都是淡黄色至浅棕色。这充分保证了在 3-4 周内次培养所需要的易碎愈伤组织。然而，随着次培养的开始，愈伤组织的生长速度会大幅度下降并且会导致愈伤组织生长缓慢并成棕色团块。据推测， 这些棕色的渗出物是酚醛树脂性质，最终能导致细胞死亡。这个普遍的现象完全是随机的，不可预测的。一旦 这个现象被触发，把 细胞被转移到新的培养基也无法挽救。然而，通过给培养基添加聚乙烯吡咯烷酮有助于保持细胞活力和生长。我们在愈伤组织培养上的经历与那些研究相似。我们的研究
39、发现那些早期（通常在 2 个星期内）开始培养的愈伤组织在次培养时不会增生，会变成棕色并且坏死。相比之下，在外植体第一次被放置到培养基上后培养 4 个星期到 4 个月的愈伤组织能够继续进行次培养，如果每间隔 1-2个月进行转移的的话。外植体中紫杉醇的存在似乎不会影响次培养后愈伤组织的存活比例。紫杉醇浓度与愈伤组织培养之间的关系还不是很清楚。C.糖的作用蔗糖是植物细胞培养中植物生长首选的碳源，尽管像葡萄糖这种代谢更加迅速的糖的存在利于快速增长。其他的糖，例如乳糖，半乳糖，果糖也能在一定程度上促进细胞生长。另一方面，糖醇，比如甘露醇和山梨(糖)醇， 一般用于提高培养基的渗透力，一点也不能促进生长。所
40、添加的糖的浓度对紫杉醇的生产发挥重要作用。一般来说，提高初始糖的浓度会导致次生代谢产物的生产增加。高浓度的糖会增加培养基的渗透能力，尽管渗透力对次生代谢的作用还不清楚。基姆以及他的同事证明最高级别的紫杉醇获得果糖。生产紫杉醇的每种糖的最高浓度在所有情况下都是 6。Wickremesinhe 和 Arteca 提供了额外的证明支持果糖对紫杉醇生产的作用最大。然而，糖的其他结合，如蔗糖与葡萄糖的结合也能增加紫杉醇生产。 细胞转化和快速的外蔗糖水解的存在已在许多细胞培养中观察到。这些报告表明细胞表面的细胞分泌物超过转化量会使蔗糖水解的速度更快。在细胞早期生长阶段蔗糖的水解加上葡萄糖的快速利用增进了细
41、胞后期生长过程中培养基中果糖的可利用性。增加果糖利用的这个时期与细胞的快速生长阶段一致。D. 毒莠定和茉莉酸甲酯的作用毒莠定能提高生长速度，而据报道茉莉酸甲酯在紫杉醇和其他紫杉烷的生产中是一种有效的刺激素。然而，我们对它刺激这些次生代谢产物的机制或途径知之甚少。毒莠定被 Furmanowa 及其同事， Ketchum 以及 Gibson 所使用，但他们没有给出毒莠定对生长速度的影响的细节。Furmanowa 及其同事在有光和无光的条件下观察了愈伤组织的生长。在黑暗中生长状况最佳的愈伤组织显示了 9.3 倍的增长，而在有光的条件下仅增长了 2-4 倍。在不加毒莠定的条件下，愈伤组织增长 0.9
42、倍。不幸的是，这种生长素对紫杉烷的生产没有影响，并且愈 伤组织快速增长速度很不稳定。当给植物运用外部刺激时，茉莉呈现出不同的形态和生理活动。他们诱导参与形成次生代谢产物的基因的转录激活。茉莉酸甲酯被证明能促进愈伤组织和悬浮培养中紫杉醇和西双版纳粗榧碱的生产。然而，与 B5 培养基相比，紫杉 烷生产选择用 WP 培养基是最好的。这可能是由于 WP 培养基硝酸钾的浓度低以及缺少硫酸盐的缘故。E. 硫酸 铜和氯化亚汞的作用在植物细胞培养中，金属离子被证明对改变次生代谢产物的运输途径有很大的影响。次生代谢产物，如萜烯类物质， 由含有 氯化亚汞的甘薯根组织产生。然而硫酸铜的影响尚未报导出来。F.生长动力
43、学和紫杉醇生产紫杉醇的低产量可能归因于尚未被完全了解的紫杉烷生产动力学。一些报导陈述了紫杉烷生产动力学的不确定的结果。为了测定紫杉烷生产的数量，我们需要进行更多的研究。据 Nett-Fetto 所说，紫杉醇生产的最大瞬时速率发生在细胞繁殖的第三个星期。紫杉醇产量不是下降就是没期望进一步培育增长。紫杉醇生产对培养基条件的轻微变化都很敏感。由于这种敏感性，细胞维持的条件，尤其是起始的细胞密度，次培养的间隔时间长度和温度必须尽可能保持准确。最近，Byun 及其同事针对细 胞生长动力学和紫杉烷 生产进行了一次很详细的研究。在他们的调查中，据观察细胞最重的时候是繁殖后的第七天。类似的，10-双乙 酰浆果
44、赤霉素和浆果赤霉素浓度最高的时候据检测分别是在第 5 天和第 7 天。 这个结果表明他 们是紫杉醇的代谢中间体。然而，紫杉醇的 最高浓度据检测是在第 22 天但会逐渐减少。Byun 及其同事表明紫杉醇可能是像 10-双乙酰浆果赤霉素和浆果赤霉素这样的代谢中间体，或者是由于细胞形态的变化或细胞死亡的 DNA 降解特性使紫杉醇进行了分解。Pestchanker 的 团队通过比较在摇瓶中的悬浮培养和对空气进行搅拌和混合的威尔逊型反应堆中的悬浮培养对紫杉醇生产动力学进行了研究。这个研究得出的结论是这些东北红豆杉的培养能在 3 周内产生大量紫杉醇（每天 1.1mg/L）。它也表明摇瓶悬浮培养和威尔逊型反应堆悬浮培养都能生产同样的紫杉醇量。然而，威尔逊型反应堆悬浮培养能更快地吸收营养（例如糖，磷酸盐， 钙和硝酸盐）。这很可能是由于威尔逊型反 应堆中生长环的存在。因此，通 过威尔逊型反应堆悬浮培养的生长速度只有 135 mg/L，而 摇瓶悬浮培养在 3 周内增长到了 310 mg/L。检讨过去，我们知道严格控制培养条件对紫杉醇的连续生产是必要的。培养基成分的轻微改变就会对细胞的生理状况产生重大影响，从而影响产品的增长和形成。为了保持细胞活力和稳定性，所有影响植物细胞生长和生产的操作必须谨慎地加以综合控制。
