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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 Production Several alternative sources of paclitaxel have been identified and

2、are 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 antin

4、eoplastic 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 wo

5、uld 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

6、method 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.

7、 The 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 medi

8、a 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. In

9、itiation 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

10、/L 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 foll

11、owing 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

12、 triggered, 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 (

13、usually 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. Th

14、e presence 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 rapi

15、dly 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 th

16、e production 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 colleagu

17、es 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

18、production. 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

19、or possess 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 coi

20、ncided with the faster growth phase of the cells.D. Effect of Picloram and Methyl Jasmonate Picloram (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 i

21、s known 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 prese

22、nce and 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 v

23、ery unstable. 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 de

24、rivative) 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

25、Chloride Metal 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 sulfa

26、te, however, 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 ord

27、er to quantitate 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 se

28、nsitive 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 c

29、ell growth 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 meta

30、bolic intermediates 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 cellu

31、lar morphological 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

32、cultures of 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 (

33、i.e. sugars, 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 control

34、led culture 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

35、be carefully 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

36、的可能性上。 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

37、组织生长。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充了1-2mg/L 2，4-二氯苯氧乙酸的B5培养基中

38、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，

39、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

40、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