【作者】 龚彦文；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2006， 博士

【摘要】 为了研究剪切力对红豆杉细胞一氧化氮(NO)和促分裂原活化蛋白激酶(MAPK)的影响,在膜式Couette反应器中悬浮培养东北红豆杉细胞,研究了剪切力作用下细胞内NO和氧化还原态的变化、H2O2和MAPK调控模型的稳定性,试图从调控模型的稳定性角度分析细胞信号的动态特征。研究了剪切力作用下红豆杉细胞中NO的变化及其作用。实验发现剪切力作用下红豆杉细胞中NO的浓度在5h达到最大,NO和SNP等氮氧化物抑制了红豆杉细胞谷胱苷肽转硫酶(GST)的活性,GST活性降低有利于保持细胞内GSH的含量和延缓细胞内微环境趋于氧化态。用活性氧的代谢模型定量地分析了NADPH在抗坏血酸-谷胱苷肽(ASC-GSH)循环和活性氧清除中的作用,结果表明细胞内的ASC和GSH被用于清除活性氧,NADPH是维持ASC-GSH循环所必须的还原剂,NADPH缺乏引起细胞内ASC、GSH氧化和细胞内H2O2的浓度增大。引入比例、积分和微分调节概念,把H2O2的产生和清除过程分别看作信号的比例放大和积分或微分反馈调节过程,用Simulink模拟了不同调控模型中H2O2的动态特征,结果显示积分负反馈调节模型与H2O2的实验结果相吻合。探讨了MAPK调控模型的稳定性,负反馈Huang-Ferrell模型和Kholodenko模型是一致的,两种模型都存在双稳态区和磷酸化MAPK的振荡区,增大MAPK浓度或磷酸化速率都能引起负反馈Huang-Ferrell模型中MAPK的振荡。激发MAPKKK磷酸化的酶E1的周期性变化也能产生与上述两种模型类似的振荡特征。多分辨分析方法能在二维空间上区分不同条件下H2O2和磷酸化MAPK的动态差别。对比了传统式和膜式Couette剪切反应器中溶解氧的差异,结果表明膜式反应器中能维持较高的溶解氧,在相同的实验条件下,膜式反应器中的细胞活力较高。通过考察剪切力作用下红豆杉细胞壁和细胞壁蛋白的变化,发现活性氧引起胶质的交联反应和蛋白表达的差异,随着剪切力时间延长,交联反应程度和细胞壁中蛋白质分子总的无规则卷曲程度都增加,同时细胞壁中WAK1的表达也增加。用主成分聚类法分析了剪切力作用前后细胞壁的差异,发现剪切力作用8h后,细胞壁的差异才明显。更多还原

【Abstract】 The effects of shear stress on nitric oxide (NO) and mitogen-activated protein kinase (MAPK) in Taxus cuspidata cells were investigated in a membrane-Couette. The changes of NO and cell redox, the robustness of H2O2 and MAPK regulating models in Taxus cuspidata under shear stress were investigated. We attempted to analyze the dynamic characteristic of signal regulating models by the model robustness.Changes and the physiological role of NO in Taxus cuspidata under shear stress were investigated. NO concentration in cells reached maximum 5h after shear stress. NO and other nitrogen species inhibited the glutathione S-transferase (GST) activity. The suppression of GST was useful for keeping the GSH content and cell redox. The role of NADPH in ascorbate- glutathione (ASC-GSH) cycle and in AOS elimination was analyzed quantificationally by the metabolic model of AOS elimination. The results showed that ASC and GSH were depleted in the early stage under shear stress. NADPH was required for AOS elimination for long stress. The cell redox would turn to oxidized state while ASC and GSH were oxidized since NADPH was depleted.The proportion, integration and differentiation regulating conception were used in H2O2 regulating model. The genaration and elimination process were regarded as proportional and integral or differential regulation, respectively. The dynamic change of H2O2 was simulated by Simulink using different model. The results showed that the negative feedback model with integrator was according with the experiments of H2O2. The robustness of the MAPK model was analyzed. Huang-Ferrell model with negative feedback control was consistant with the Kholodenko model. The two models have double steady-state respectively and the oscillation property was occurred at special range. The oscillation occurred while the reactive rate of MAPKKK phosphating or MAPK concentration was increased. Periodical change of E1 which activated MAPKKK phosphating caused oscillation in MAPK-PP, which was similar to the above models. The multiresolution analysis was used to discriminate the dynamic properties of H2O2 and MAPK-PP. The dynamic characteristics at different conditions were distinguished at the two-demensions.The dissolve oxygen was compared between conventional type and membrane-Couette. The results showed that the oxygen concentration cell viability更多还原