【作者】 杨松；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2007， 博士

【摘要】 为了更好地研究红豆杉细胞诱导凋亡过程及细胞自发凋亡过程的生理和生产特性,本文以不同诱导子胁迫培养下红豆杉细胞及不同品系红豆杉细胞作为研究体系,运用高通量分析策略并结合生物信息学及信号转导研究思想,试图探究细胞次生代谢产物生产强化的信号机制。基于LC/ESI/MSn和ESI-MS/MS构建了南方、东北红豆杉细胞膜磷脂图谱,准确地定性、定量了PG、PE、PC、PI、PS、PA及LysoPC共7类磷脂100多种磷脂分子。引入信号转导研究策略,并结合主成份聚类手段,分析了红豆杉细胞正常生长和自发凋亡中磷脂的代谢差异,发现南方红豆杉细胞磷脂酶D(PLD)在培养17~22 d中显著的激活,水解底物PC生成信使分子PA导致细胞膜双分子层的坍塌是其自发凋亡的重要原因。以稀土(铈,Ce4+或Ce3+)和植物激素(茉莉酸甲酯,MeJA)为例,研究了诱导子胁迫下红豆杉细胞膜磷脂的代谢调控。实验发现Ce4+导致细胞磷脂总含量在48~72 h明显减少,而MeJA诱导0~72 h磷脂总量没有变化。主成份聚类法分析36个脂质提取样本,揭示了Ce4+诱导的凋亡细胞和Ce3+、MeJA诱导的非凋亡细胞及正常培养细胞的区分度达到82.7%,正交偏最小二乘法预测了8种PA、2种LysoPC和10种PC是潜在的脂质生物标志物。对比了Ce4+和MeJA激活PLD及磷脂酶A2(PLA2)的启动时域及其信号功能,发现PLD和PLA2在时序、程度上差异激活是生物标志物产成的主要原因。MeJA诱导PLD 5~14 h适量激活,而Ce4+诱导PLD 2~24 h显著激活,产生高浓度PA信使分子激发了下游凋亡途径,表明不同浓度的PA在防御响应中具有不同的调控机制。MeJA微弱激活PLA2,而Ce4+诱导PLA2 1~4 h高水平激活,释放LysoPC和自由脂肪酸,强化胞内茉莉酸(JA)不断积累。PLA2抑制剂实验提出JA途径激活是Ce4+强化紫杉醇生产的必要前提之一,但JA途径并不直接调控凋亡通路。探讨了细胞诱导凋亡中类ERK激酶激活和O2·-迸发的变化规律,发现这两个信号事件在早期(0~2 h)应是相互独立的,NADPH酶抑制实验揭示诱导4 h后O2·-迸发对类ERK激活有明显信号负调控作用。以Ce4+诱导体系为例,初步构建类ERK途径、O2·-迸发途径及脂质信号途径之间相互协调的生物学模型。更多还原

【Abstract】 In order to well-understand the growth and production of Taxus cells during the elicitors-induced apotposis and cellular spontaneous apoptosis, we investigated series changes of signal molecules by using high-throughput technologies, signal transduction strategies as well as bioinformatic methods. From the work we gained some valuable results.A lipidomic approach was employed to investigate the changes in membrane glycerophospholipids during the procession of cellular development and apoptosis of two plant cell lines, Taxus cuspidata and Taxus chinensis var. mairei. Analysis of lipids by LC/ESI/MSn and ESI-MS/MS coupled with principal component analysis profiled 7 glycerophospholipid classes and more than 100 glycerophospholipid molecular species, and indicated significant differences in the abundance of glycerophospholipids. Continuous increase of phospholipase D (PLD) activity from 17 day to 22 day led to PA production in apoptotic T. chinensis var. mairei cells suggesting that PLD activation and PA formation mediated spontaneous apoptosis.Markedly decreased levels of total glycerophospholipids were observed at 48 h and 72 h following treatment with cerium (Ce4+). The total glycerophospholipids levels, however, were not significantly altered for methyl jasmonate (MeJA)-induced T.cuspidata cells. Using multivariate analysis, especially orthogonal partial least squares (OPLS), we discovered that LysoPC, PA and PC were three important lipid groups that were responsible for the discrimination between MeJA/Ce3+-induced non-apoptotic cells and Ce4+-induced apoptotic cells.Continuous activation of PLD from 3 to 24 hour led to PA production during Ce4+-triggered apoptotic response, whereas a delayed and attenuated induction was observed during MeJA-triggered non-apoptotic defense response. PA can act as a mediator in two opposite ways, and appropriately high concentrations of PA were important determinants of apoptosis in Ce4+-induced T.cuspidata cells. Rapid increase of phospholipase A2 (PLA2) activity (1-4 h) caused a massive production of LysoPC following Ce4+ elicitation. In contrast, PLA2 activity was poorly induced following MeJA elicitation. PLA2 inhibitor suppressed not only jasmonic acid (JA) accumulation but also taxol production in Ce4+-induced cells, suggesting that PLA2 activation mediated taxol production partially via a JA-dependent signaling pathway. In addition, JA signaling pathway was not linked directly to apoptosis of T. cuspidata.An approximately 46 kDa ERK-like MAP kinase appeared to be activated at 5 minutes in Ce4+-induced T. cuspidata cells. It was possible that ERK-like activation and O2·- burst were separate signal events in early. O2·- burst formed a signal negative regulation to ERK-like activity. The present study suggested that down-regulation of ERK-like MAPK, burst of O2·-, activation of phospholipase and generation of lipid molecules as the key signaling pathways mediated apoptosis and taxol biosynthesis in Ce4+-induced T. cuspidata cells.更多还原