【作者】 雷晓勇；

【导师】 戴尧仁；

【作者基本信息】 清华大学 ， 生物学， 2003， 博士

【摘要】 我们利用抗氰呼吸途径末端氧化酶AOX(alternative oxidase)的单克隆抗体研究了抗氰呼吸与植物细胞凋亡之间的关系,发现在乙烯诱导的番茄原生质体的凋亡过程中,AOX的表达明显提高;通过抗氰呼吸途径的电子传递也大大增加。另外,当用抗氰呼吸途径的抑制剂SHAM(salicydroxamic acid)预处理番茄悬浮细胞1小时后,再用乙烯诱导凋亡,发现TUNEL阳性反应和DNA laderring明显加速,说明抗氰呼吸途径不但参与了乙烯诱导的细胞凋亡,而且还起着抑制凋亡的作用。在进一步的实验中发现,位于细胞内膜上的磷脂酰丝氨酸(phosphatidylserine,PS)在乙烯诱导过程中向膜外翻转、线粒体膜膜电位(ΔΨ m )降低、类caspase-3酶活性升高以及PARP的剪切激活等典型的细胞凋亡特征出现。然而,同样是凋亡细胞主要特征的位于线粒体细胞色素电子传递链末端的Cyt c的释放却没有发生。实验结果表明,抗氰呼吸可能在其中起着重要作用,它的启动可以在一定程度上减少ROS的产生。在动物细胞中,褪黑激素能够通过清除体内ROS来抑制细胞凋亡。我们采用TUNEL检测、DNA laddering分析以及电子显微镜观察等方法发现,用10-20ng/mL褪黑激素预处理胡萝卜悬浮细胞可以显著地减轻低温(2-3℃)诱导的细胞凋亡。在动物细胞中,褪黑激素的抗凋亡作用往往都归功于其快速而高效的清除自由基功能。然而,在胡萝卜细胞中,褪黑激素的抗凋亡作用却不是依靠这一功能,因为我们并没有发现细胞内自由基发生明显的变化。利用高压液相色谱法发现多胺含量明显增加,特别是腐胺和亚精胺。另外,我们用外源多胺代替褪黑激素预处理胡萝卜悬浮细胞,发现两者对低温诱导的细胞凋亡有着相同抑制效果。说明多胺很有可能参与了褪黑激素对低温诱导的胡萝卜悬浮细胞的抑制过程。更多还原

【Abstract】 In this study, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to detect the expression of alternative oxidase (AOX) protein in tomato mitochondria. The results showed that there was an obvious correlation between the ethylene-induced apoptosis and the levels of AOX protein in tomato cells undergoing ethylene-induced apoptosis. In addition, when tomato protoplasts were preincubated with 2 mM salicylhydroxamic acid (SHAM), an inhibitor of alternative pathway, before being exposured to ethylene, the TUNEL positive reaction and DNA fragmentation was obviously accelerated. We suggest that AOX may play an important role in protecting tomato protoplasts against ethylene-induced apoptosis in tomato protoplastsThe cleavage of poly (ADP-ribose) polymerase (PARP) and activation of caspase-3-like proteases in ethylene-induced apoptosis of tomato protoplasts were investigated. Our results showed that after a 4 h treatment with ethylene, PARP was cleaved into an 84 kDa fragment, while DNA laddering appeared only after a 12 h treatment. At the same time, an activation of caspase-3-like protease was observed. Furthermore, when tomato protoplasts were preincubated with 2 mM 3-AB or 4 mM NIC, the specific inhibitor of PARP, before treatment with ethylene, apoptotic cell death was reduced significantly. Taken together, our results indicated that the cleavage of PARP and activation of caspase-3-like cysteine protease are probably involved in the progression of apoptosis in ethylene-induced apoptosis.Pretreatment with 10 ng/mL-20 ng/mL melatonin for 5 days significantly attenuated cold-induced apoptosis in carrot suspension cells (Daucus carota L.) as evidenced by the TUNEL procedure, DNA fragmentation and the <WP=6>morphological changes revealed by electronic microscopy observations. The antiapoptotic effect of melatonin was initially thought to be a result of its antioxidant actions. In our study, however, reactive oxygen species (ROS) generation was found not to be affected by melatonin treatment, suggesting that melatonin plays its protective role not related to its direct ROS scavenger. At the same time, notable increases in putrescine and spermidine levels were observed in melatonin-treated cells, which may be responsible for the alleviation of the cold-induced apoptosis. The possible involvement of polyamines in antiapoptotic effect of melatonin was further confirmed by the inhibitory effect of exogenous polyamines on apoptosis as displayed by the DNA laddering assay.更多还原