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微囊藻毒素对高等植物的毒理效应及作用机制研究
Alternative TitleStudies on the Toxicological Effects of Microcystins against Higher Plants and the Toxicity Mechanisms
黄文敏
Subtype博士
Thesis Advisor刘永定
2009-06-03
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword微囊藻毒素 烟草 悬浮细胞 营养生理 超微结构 线粒体 凋亡 坏死
Abstract蓝藻水华的频繁暴发已经成为一个在世界上有广泛影响的生态灾难。蓝藻水华污染所带来的主要危害之一是产生蓝藻毒素并造成生态影响,包括损害人体健康和伤害其它生物。在已发现的各种蓝藻毒素中,微囊藻毒素的危害最普遍,其毒性较大,已有的研究最多。以往有关微囊藻毒素的毒性研究大多数集中在动物上。高等植物-生态系统的主要初级生产者,其种类的多样性和初级生产量直接影响生态系统的结构和功能,目前国内外也陆续开展了微囊藻毒素对高等植物的毒理学研究,但是研究还不够深入,微囊藻毒素对植物的具体毒性机制还未得到充分的阐明。本文选择模式植物烟草(Nicotiana tabacum L.)的悬浮细胞为实验材料,研究了微囊藻毒素对其的毒性效应及作用机理。主要进展和研究结果如下: 1.微囊藻毒素的制备 以取自滇池的微囊藻干粉为材料,采用常规的制备方法,大量制备并获得了一定纯度的粗毒素,经HPLC分析,粗毒素主要为MC-RR。MC-RR纯品是粗毒素经制备型HPLC进一步纯化获得,经HPLC检测分析,含量达95%以上,可用于常规的毒理学实验。 2.微囊藻毒素诱导烟草细胞氧化胁迫的进一步论证 分别用两种外源抗氧化剂DMSO和ASA协同MC-RR处理烟草细胞,观察加入外源抗氧化剂后细胞内ROS和抗氧化系统的变化。外源抗氧化剂的加入,使胞内ROS及MDA含量恢复至对照水平,抗氧化系统的各个指标也恢复到对照水平。表明,一定浓度的外源抗氧化剂可以保护烟草细胞,减少细胞内ROS的积累并降低细胞的氧化应激反应,这更进一步揭示了微囊藻毒素的毒性机制是与诱导氧化胁迫密切关联的。 3. 微囊藻毒素影响烟草细胞活力,并降低了细胞的正常生理态势 用不同浓度(0.1、1、10 µg/mL)的MC-RR处理烟草悬浮细胞,测定了细胞活力及与营养生理代谢相关的指标。结果表明,高于0.1 µg/mL的MC-RR处理2d后显著降低烟草细胞的活力,并减少细胞内蛋白质的含量,暴露4d后细胞吸收氮、磷的能力明显下降;细胞活力到处理后期有所恢复,可能是细胞逐渐适应了毒素的胁迫。0.1 µg/mL MC-RR处理引起细胞内可溶性糖含量上升,而10 µg/mL MC-RR处理导致细胞内可溶性糖含量下降。可溶性糖含量的不同变化趋势反映了烟草细胞对不同浓度的MC-RR有不同的响应能力。可溶性糖合成增多,烟草细胞表现出主动适应,通过合成较多能量物质以抵抗毒素的胁迫;而当毒素浓度过高,进入细胞内的有效毒素浓度也随之增多时,烟草细胞需要不断消耗能量来维持细胞基本的生理功能。上述结果表明,高于0.1 µg/mL的MC-RR暴露在一定程度上降低烟草细胞的正常生理态势。 4. 微囊藻毒素引起烟草细胞特征性形态及超微结构改变,暴露浓度不同引起改变的程度有异 60 µg/mL MC-RR处理烟草细胞5天后,DAPI荧光染色观察到细胞核染色质高度浓缩,呈新月形在核膜边缘聚集;电镜观察结果显示:细胞质壁分离,线粒体轻微水肿,内质网朝线粒体方向涡旋,细胞核收缩,核内染色体高度浓缩并在核膜边缘聚集。超微结构的改变以及DAPI染色观察到的细胞核的变化符合典型的细胞凋亡的特征。此外,还观察到大量小泡和溶酶体,溶酶体内还可见结构清晰的线粒体残体,这是细胞自我吞噬现象的表征。自噬作用可为细胞提供一种保护机制,赋予细胞一定的耐受MC胁迫的能力。120 µg/mLMC-RR处理后的烟草细胞,与对照和60 µg/mL MC-RR处理组相比,细胞形态和超微结构发生了显著的改变:细胞散在排列,零星聚集;细胞膜上多处破裂,并呈溶解弥散状态;细胞器严重肿胀。随着处理时间的延长,细胞的肿胀,细胞质膜和核膜的破裂更加明显,胞内内含物也释放到周围环境中。这些结构的改变十分符合细胞坏死的结构特征。作者在两组毒素处理组中还发现了大量小泡,且小泡内含有不规则的沉积物。作者推测这些沉积物是MC与谷胱甘肽结合的络合物,被运输至封闭小泡内,将其限制在固定的区域,使其无法在细胞质内自由流动,将毒素对细胞的伤害尽可能减到最低。本研究首次从超微结构角度论证了不同剂量的微囊藻毒素对植物细胞的毒性方式不同,并且细胞自身也通过调节超微结构以适应毒素的胁迫。 5. 微囊藻毒素导致胞内线粒体轻微肿胀、功能受损,可能是线粒体内活性氧产生与清除失衡所致 10 µg/mL MC-RR处理烟草细胞后,诱导了烟草细胞线粒体PT孔不可逆开放,线粒体膜电势降低,线粒体膜上的Ca2+-ATP酶、H+-ATP酶和Na+-K+-ATP酶活性均被诱导升高;线粒体超微结构同时发生一定的损伤,部分线粒体异常肿胀,并且在线粒体基质内观察到髓鞘样结构。检测线粒体ROS产生速率发现其显著高于对照,表明线粒体内的ROS产生与清除失去平衡。从线粒体结构及特征性的功能指标的检测结果来看,MC-RR造成的线粒体损伤可能与ROS有关。 6. ROS和线粒体PT孔、呼吸链参与了微囊藻毒素诱导烟草细胞凋亡的信号转导过程,并且毒素诱导细胞凋亡必需caspase-3/-9的活性表达 通过系列实验建立了60 µg/mL MC-RR诱导烟草细胞凋亡的模型,在此模型中引入流式细胞术进行准确的定量分析。研究发现,60 µg/mL MC-RR诱导烟草细胞凋亡的过程中,ROS的累积是影响凋亡的关键环节-外源抗氧化剂ASA预处理后,削弱了胞内ROS的累积但同时细胞凋亡率也显著降低;线粒体PT孔的开放状态也是影响凋亡的关键因素,当使用PT孔的特异性抑制剂CsA预处理后,PT孔开放受到抑制,线粒体膜电势得以回升,细胞的凋亡率也明显下降,可见线粒体PT孔的开放状态影响细胞凋亡。深入研究发现,线粒体呼吸链复合物Ⅰ、Ⅲ是线粒体活性氧产生的主要部位,且复合物Ⅰ、Ⅲ的抑制剂在抑制ROS生成的同时也降低了细胞凋亡率。可见,线粒体呼吸链参与了凋亡过程,并且为MC-RR、ROS累积和细胞凋亡三者之间提供了一个连接的桥梁。研究还发现,毒素诱导细胞凋亡的模型中,caspase-3、9活性显著升高;而当使用caspase-3的特异性抑制剂AC-DEVD-CHO处理细胞后,细胞凋亡率显著下降,表明caspase-3的活性表达是毒素诱导细胞凋亡发生的必需条件。 7. 烟草细胞内GSH系统对MC-RR胁迫的响应 考虑到GSH在植物抗逆响应中占据的重要地位,作者研究了毒素处理烟草细胞后,胞内GSH及其相关代谢酶类的变化情况。结果表明中高浓度(1、10 µg/mL)毒素处理细胞后,胞内GSH含量在初始4天显著升高,6天后,GSH含量又下降至对照水平。tGSH含量的显著增加可以证明GSH含量的升高主要来源于新的GSH的合成。高浓度毒素处理细胞4天后,GSH/GSSG比值明显高于对照,这与细胞内充足的GSH供应是分不开的,表明细胞处于活跃的抗氧化状态,细胞内氧化应激水平增强;到第6天时,GSH/GSSG比值减小,这是胞内GSH含量下降GSSG含量升高直接导致的,提示细胞的抗氧化能力下降,细胞处于易受氧化损伤的状态。表明毒素诱导的氧化压力已经将氧化还原的平衡态推到了氧化态。对与GSH代谢密切相关的三种重要酶类:GST、GPX、GR的研究结果显示,在处理的第4天,GST、GPX和GR活性在中高浓度处理组中显著升高。以上结果表明,GSH及其代谢相关酶类都参与了烟草细胞对MC-RR胁迫的响应,GSH库水平的维持对细胞抵抗胁迫具有特殊而重要的意义。
Other AbstractToxic cyanobacterial blooms in fresh water bodies have been becoming a kind of ecological disaster worldwide. One of the most harmful effects of cyanobacteria blooms is typically from the toxins produced by the algae, which present a major threat to the health of human, livestock and wildlife. Of all the algal toxins, microcystins are the most widely distributed toxic and abundant species. Most investigations into the toxicity of microcystins are focused on animals. In this paper, the toxicological effects of microcystins on terrestrial higher plants, Nicotiana tabacum L., and its possible mechanism were studied. The main results are as follows: 1. Microcystins from Microcystis bloom in Lake Dianchi were extracted and purified with high performance liquid chromatography (HPLC). MC-RR reagent with the purity over 95% was finally obtained by preparative HPLC. 2. When tobacco BY-2 suspension cells were exposed to 2 µg/mL MC-RR with 0.5% DMSO or 2 mmol/L ASA, the formation of ROS and MDA were prevented, and the contents of GSH, ASA and activities of SOD and POD all decreased in contrast to MC-RR treatment. It is indicated that tobacco BY-2 suspension cells suffered oxidative stress after MC-RR treatment and the exogenous ASA and DMSO can protect the cells from MC-RR induced oxidative stress. 3. Adopting 0.1, 1, and 10 µg/mL microcystin-RR (MC-RR) to treat tobacco BY-2 suspension cells, the cell viability, contents of protein, soluble sugar, nitrate nitrogen (Nitrate-N), and total phosphorus were determined, and also the activity of acid phosphatase (ACP) was detected. The results showed that the cell viability and protein content were markedly decreased in both the 1 and 10 µg/mL exposure groups after 2 d compared with the control. The soluble sugar content significantly decreased in the 10 µg/mL treated cells to a value 45.57% of the control, while the soluble sugar content was elevated in the later period of low concentration MC-RR exposure. After 4 d exposure, a significant decrease in the nitrate-N content was only observed in the 10 µg/mL treatment group, while a further reduction in nitrate-N content was observed in all the treated groups after 7 d treatment. MC-RR also decreased the total phosphorus content and after 9 days exposure the total phosphorus content in 0.1, 1, and 10 µg/mL MC-RR treated cells accounted for 74.98%, 76.47% and 84.00% of control, respectively. ACP activity in 3 treated groups first decreased and then increased compared with the control. It can be concluded that when the toxin concentration was over 1 µg/mL, the nutritional metabolism were significantly restrained by MC-RR. 4. Tobacco BY-2 cells were exposed to microcystin-RR (MC-RR) at two concentrations, 60 and 120 µg/mL, to study the changes in morphology and ultrastructure of cells as results of the exposure. Exposure to the lower concentration for 5 d led to typical apoptotic morphological changes including condensation of nuclear chromatin, formation of a characteristic ‘half moon’ structure, and cytoplasm shrinkage and decreased cell volume, as revealed through light microscopy, fluorescence microscopy, and transmission electron microscopy respectively. Exposure to the higher concentration, on the other hand, led to morphological and ultrastructural changes typical of necrosis, such as rupture of the plasma membrane and the nuclear membrane and a marked swelling of cells. The presence of many vacuoles containing unusual deposits points to the involvement of vacuoles in detoxifying MC-RR. Results of the present study indicated that exposure of tobacco BY-2 cells to MC-RR at a lower concentration (60 µg/mL) results in apoptosis and that to a higher concentration (120 µg/mL), in necrosis. 5. Tobacco BY-2 cells were exposed to 10 µg/mL MC-RR. PTP, mitochondrial permeability transition pores, was studied and a time-dependent opening was observed, the loss of mitochondrial transmembrane potential (ΔΨm) was also induced. Changes in activities of Ca2+-, Na+-K+- and H+-ATPase on mitochondrial membrane of tobacco BY-2 cells were studied. Results showed that all these three ATPases increased under the treatment. Mitochondrial morphological changes were observed by TEM. The mitochondria in untreated cells had high electronic density with abundant and acerose cristae. In the MC-RR treated group, the mitochondria were slightly swollen, with lower electronic density and fewer cristae than those in the control group. These results suggested that the toxicity of microcystin-RR caused the damage in mitochondria of tobacco BY-2 cells. 6. When tobacco BY-2 cells were treated with MC-RR of 60 μg/mL for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 μg/mL MC-RR induced rapid apoptosis. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial transmembrane potential (ΔΨm) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, reversed the decrease in ΔΨm and subsequent cell apoptosis, indicating a critical role of ROS in MC-RR-induced loss of ΔΨm and apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of ΔΨm, as well as cell apoptosis, suggesting that PTP is involved in 60 μg/mL MCRR-induced tobacco cell apoptosis signalling process. Thus, it could be concluded that the MC-RR-induced ROS formation may lead to the opening of PTP, inducing the decrease of ΔΨm and subsequent apoptosis in tobacco BY-2 cells. Furthermore, it was found that MC-RR caused activation of caspase 3 and 9. Caspase-3 inhibitor AC-DEVD-CHO significantly prevented apoptosis of tobacco BY-2 cells at the 5th day, suggesting that MC-RR induced apoptosis mainly through caspase-dependent pathway. Based on the results using mitochondrial inhibitors, it is suggested that complexs Ⅰand Ⅲ of respiratory chain are potentially involved in MC-RR induced ROS generation in mitochondria. The finding of our study that MC-RR induced tobacco cell apoptosis was abrogated by mitochondrial inhibitors such as Rot and AA supports the hypothesis that mitochondria provide a link between MC-RR, ROS generation, and tobacco cell apoptosis. 7. This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 µg/mL) for 0-6 d resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 d exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 d exposure. The GSH/GSSG ratio was much higher than control in 10 µg/mL MC-RR treated group at day 4, but after 6 d exposure, the ratios in all treated groups were lower than that of the control group. It can be concluded from all the data that the inherent GSH-involved detoxifying mechanism might constitute the first line of defense against MC-RR stress. The GSH synthesis would be significantly induced and GSH-related enzymes such as GST, GR and GPX would be activated to compensate the defensive system.
Pages160
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12440
Collection学位论文
Recommended Citation
GB/T 7714
黄文敏. 微囊藻毒素对高等植物的毒理效应及作用机制研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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