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。     

培养基成分对东北红豆杉细胞生长和紫杉醇产量的影响

在东北红豆杉（taxus cuspidate)细胞培养物的基本培养基中加入30mg.L^-12.4-D,可以提高细胞培养物的生长量,其相对最高生长量可以达到85．999mgFW/gFW.d;培养基中加入活性炭粉末1g.L^-1,可以克服植物细胞生长过程中的褐化问题;适当种类及含量的生长调节剂的加入：6－BA0．5mg.L^-1＋KTmg.L^-1,可以使植物细胞合成和积累相对较多的紫杉醇（Taxol).     

东北红豆杉开发研究进展

系统论述了东北红豆杉的生物学和生态学特性,野生资源的分布现状,蕴藏量及开发前景,人工繁殖现状和技术,资源开发存在的问题及发展对策.     

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 .     

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。     

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.     

美丽镰刀菌与固定化东北红豆杉的共生培养

采用固定化培养法模拟东北红豆杉（Taxus cuspidate）和美丽镰刀菌（Fusarium mairei K178）的共生环境,对其相互作用进行初步研究以期提高各自紫杉醇产量。共培养条件为：海藻酸钠浓度 2%（M/V）,T. cuspidate细胞包埋量0.15 g/mL,CaCl2浓度4%（M/V）,凝胶直径为3～3.5 mm,钙化0.5h,以40mL接种量接入装液量为70mL/250mL摇瓶的改良MS培养基,25℃、160 r/min振荡黑暗培养,第9d接入10%（V/V）对数期的F. mairei,10d后收获。结果表明,F. mairei 对T. cuspidate细胞的生长有强烈抑制作用,能诱导紫杉醇等次生代谢物向胞外分泌;F. mairei 与T. cuspidate的相互作用对共生培养物的紫杉醇产量也有显著影响。     

东北红豆杉细胞培养生产紫杉醇的调控研究

研究了诱导子、前体及抑制剂的协调作用对东北红平杉生产紫杉醇的影响。结果表明,向培养基中加入80mg/L水到、80mg/L茉莉酸甲酯、0.5mmol/L乙酸钠、2mmol/L苯丙氨酸、0.5mmol/L丝氨酸、0.1mmol/L甘氨酸、10mg/L肉桂酸、0.5mmol/L苯甲酸钠、5mmol/L丙酮酸钠、10mg/L氯化氯胆碱、和1mg/L赤霉酸可使紫杉醇含量提高368.65%。并且证明交互作用对紫杉醇合成有显著作用。     

东北红豆杉可再生部位紫杉醇含量时空动态变化规律

利用ODS-C18反相高效液相色谱体系研究了东北红豆杉(Taxus cuspidata)叶、茎、果肉及种子中紫杉醇含量的时空动态变化。结果表明, 东北红豆杉的4个部位均有紫杉醇存在, 但存在的时间和部位具有较大差异。紫杉醇的含量最大值为0.043 mg.g-1 (FW)出现在10月份种子中, 最小值0.001 mg.g-1 (FW)出现在11月份果肉中。叶与茎中紫杉醇的含量变化较为一致, 叶和茎中紫杉醇含量在7月份达到最高, 分别为0.032 mg.g-1 (FW)和0.029 mg.g-1 (FW), 在8月份降至最低, 分别为0.011 mg.g-1 (FW)和0.010 mg.g-1 (FW)。在新叶萌发期, 老茎叶比新茎叶中紫杉醇的含量高约2.5倍。     

超声波对红豆杉悬浮细胞生长及紫杉醇释放的研究

分析了超声波对中国红豆杉悬浮细胞培养的生长,紫杉醇合成及释放的影响,细胞对不同强度及作用时间的超声波反应不同,用38kHz,120s的超声强度处理悬浮细胞,紫杉醇胞外释放率由对照的10％左右提高到40－50％,总产量提高了47％,超声波处理植物细胞,提供了在保持细胞生长的前提下有效刺激胞内次生代谢物的简易操作方法。     

Phytohemagglutinin enhancement of cell fusion reduces polyethylene glycol cytotoxicity

Phytohemagglutinin (PHA) enhances polyethylene glycol-induced mammalian cell fusion. This is reflected by an increase in the effectiveness of lower concentrations of polyethylene glycol at inducing fusion, and thereby a reduction in the cytotoxicity of the fusogen.     

Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability

In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 μM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.     

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     

Effects of divalent cations and polyethylene glycol on the membrane fluidity of protoplast

Calcium is often used to stabilize membranes and enhance membrane fusion. We have used the fatty acid spin label, 5-nitroxy stearic acid to measure fluidity changes in the plasma membrane of carrot suspension culture cell protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2A_max) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millimolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2A_max and evidence of exchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a living system describe membrane structural changes under conditions similar to those used for protoplast fusion.     

Effects of polyethylene glycol and dextrans on immunoprecipitations: a two-cross immunodiffusion study

Precipitating titers and immunochemical titers obtained in a wide range of antigen-to-antibody concentration ratios by the two-cross immunodiffusion technique are compared with the corresponding laser light scatter precipitin curves. The two-cross immunodiffusion technique has also been applied to investigate whether polyethylene glycol of molecular mass 6000 and dextrans of molecular masses from 10,000 to 2,000,000 enhance the immunoprecipitation processes of the system human serum IgG-rabbit immune serum at pH 5.5 and 8.1 at 20 degrees C. It was found that the significant increase of precipitating titers of both precipitating components in the presence of polyethylene glycol is a consequence of a strong decrease of solubility of the primary antigen-antibody complex. The decrease of solubility does not affect the immunochemical titer of the immune serum, indicating stoichiometrical invariance of the precipitate at the equivalence. The apparent strong decrease of diffusion coefficients of both antigen and antibody in 20- and 40-g/liter polyethylene glycol solution is attributed to increase of viscosity of the solutions and to a partial self-association of protein molecules due to steric exclusion. In 40-g/liter polyethylene glycol solutions at pH 5.5 every fourth molecular entity of antigen and every third molecular entity of antibody are present in the form of a two-molecular self-associate, whereas in 20-g/liter polyethylene glycol solutions only 1% of antigen molecules and 8% of antibody molecules are associated. With the increase of pH to 8.1 the self-association of protein molecules is strongly further enhanced. Dextrans in 20-g/liter solutions, without regard to their relative molecular masses, do not influence precipitating titers and solubility of the antigen-antibody system at equivalence and do not enhance self-association of protein molecules. The strong decrease of diffusion coefficients of immunoglobulin G antigen and antibodies in dextran solutions is solely attributed to the increase of viscosity of the dextran solutions; hence there was no evidence of interaction of dextrans with serum IgG proteins.     

Reexpression of HPRT activity following cell fusion with polyethylene glycol

Polyethylene glycol-1000 (PEG-1000) induced fusion of HPRT (E.C. 2.4.2.8) deficient Chinese hamster cells with -galactosidase A (E.C. 2.3.1.22) deficient cells from a patient with Fabry's disease yielded hybrids which contained both human and hamster HPRT, G6PD (E.C. 1.1.1.49), and APRT (E.C. 2.4.2.7) and Chinese hamster -galactosidase B. Thus PEG-1000 mediated somatic cell fusion led to reexpression of Chinese hamster HPRT. It did not restore the expression of human -galactosidase. Since PEG-1000 treatment of HPRT^– Chinese hamster cells in the absence of human cells yielded no HPRT⁺ cells, it is concluded that the element responsible for the restoration of rodent HPRT was contributed by the human cells and not by the agent employed to promote fusion.This work was supported by research grants from the United States Public Health Services GM 17702, from the National Science Foundation BMS 74-21424, and from the National Foundation March of Dimes 1-377.