【作者】 王雪青；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2003， 博士

【摘要】 为了研究红豆杉细胞对紫杉醇(taxol)防御反应的机制，以人工半合成的taxol类似物多烯紫杉醇(taxotere)代替taxol，建立了taxol诱导红豆杉细胞悬浮培养体系，结果表明taxol作为类诱导子激活了红豆杉细胞防御响应。通过研究不同浓度的DMSO和乙醇对东北红豆杉细胞代谢和细胞膜通透性的影响，发现一定剂量的DMSO和乙醇抑制了细胞的生长、降低了细胞活力、增大了细胞膜的通透性。在相同浓度的条件下，DMSO比乙醇对细胞的毒性小，低于1% (v/v) 的添加量，DMSO不影响细胞代谢和膜的通透性，而只是短时改变了酶的活力，而同样浓度的乙醇则会严重干扰细胞的代谢，破坏细胞膜的完整性。系统研究了taxol和taxotere对不同生长时期红豆杉细胞代谢的影响。发现在对数前期加入taxol和taxotere，可显著抑制细胞的繁殖；而在对数生长后期加入taxol和taxotere，对细胞的增殖无显著影响，说明taxol及taxotere对不同生长时期红豆杉细胞的作用机制不同。系统研究了taxol和taxotere诱导红豆杉细胞培养体系的防御响应机制。适当浓度的taxol (10 μM) 和taxotere（35 μM）可诱导细胞产生防御响应，包括明显的H+跨膜内流和活性氧积累的早期防御响应以及随后活化PAL和促进taxol合成的晚期防御响应。采用二维凝胶电泳技术，分析了taxol诱导细胞产生早期防御响应的蛋白表达差异，发现taxol诱导了10个新蛋白点的表达，其中一些新蛋白点的分子量与细胞信号分子G-蛋白、Ca2+依赖蛋白激酶的分子量接近。应用NADPH特异性抑制剂研究发现NADPH氧化酶激活是外源taxol和taxotere诱导细胞产生活性氧产生的主要机制，H2O2是活性氧积累的主要成分。系统分析了taxol和taxotere在有、无细胞培养介质中，浓度的动态变化规律和考察了通过改变细胞膜通透性taxol的释放的机制，结果显示胞内taxol的释放存在逆浓度的过程，膜通透性的增大在一定程度上促进了taxol释放，但并不是主要机制，红豆杉细胞对taxol的运输可能存在着主动运输系统。更多还原

【Abstract】 The suspension cultures of Taxus spp. induced by taxol and taxotere were established to study the defense-response mechanisms of suspension-cultured Taxus spp., using taxotere as taxol indicator. The results showed that taxol as elicitor-like stimulated defense responses.The effects of DMSO and ethanol, used for dissolving taxol or taxotere, on the metabolisms and membrane permeability of Taxus cuspidata cells in the suspension cultures were studied to lessen themselves toxicity to the cells. It was found that DMSO and ethanol over some additives levels inhibited cell growth, decreased the cell viability, increased the cell membrane permeability, and that DMSO was less poisonous to cells than ethanol at the same level. DMSO below 1% (v/v) did not affect cell metabolism and membrane permeability of Taxus spp. cells, only altered enzyme activity related ROS and alkalization of medium in reverse short-time manner. But ethanol in additional of the same level significantly influenced cell metabolism and membrane permeability. So 1% (v/v) DMSO is an ideal solvent and additive amounts. The effects of taxol and taxotere with various concentrations on the suspension cultures in different growth stage were investigated. The results showed that taxol and taxotere remarkablely inhibited the cell growth at the early exponential phase of suspension cultures but did not affect at the late one. So a novel viewpoint was first put forward that the functions of taxol (10 μM) and taxotere（35 μM）on the cells at late exponential phase were as a non-specific elicitor. The defense responses mechanism of Taxus spp. cells to taxol or taxotere were systemic studied, on the base of investigating the extracellular pH and ROS accumulation for the early defense responses and determining PAL activity, taxol concentration, protein contents for the late defense responses. The results showed the appropriate concentration of taxol (10 μM) or taxotere (35 μM) activate the influxes of H+, ROS accumulation, PAL activity and biosynthesis of taxol, as well as apoptosis, however, the high concentration taxol or taxotere (140 μM) did not show above results. Ten plots of protein were only observed in taxol-induced cells and there were differences at expression level of some plots between control and taxol-induced cells using two dimension electrophoresis, which reflect those differential protein were highly relevant to signal moleculars of cell defense responses, suggesting cell <WP=4>apoptosis and taxol biosynthesis might be parallel or two events in the same pathway of defense responses.With application of pharmacological inhibitor and of determining the changes of enzymes of CAT, POD, SOD, the source and main composition of ROS were studied. The results showed that taxol (10 μM) and taxotere (35 μM) could reduced ROS accumulation and that NADPH oxidase was the main mechanism of ROS accumulation , H2O2 is the leading component of ROS. Taxol in the cells, which pre-incubated with exogenous, released into fresh medium was studied with application of solvents altering membrane permeability of Taxus spp. cells, The results showed that increasing membrane permeability promoted release of intracellular taxol to some extent. It is first supposed that the active transport system of taxol in Taxus cells be exist, by comparing and analyzing the changes of taxol or taxotere in cells or free-cells systems.更多还原