【作者】 邢鸣鸾；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 近年来,随着人类活动的不断增强、社会经济的快速发展,含有大量生物所需的氮、磷等营养物质的城市生活污水和工农业废水被超负荷排放入湖泊、水库和海湾等多种流速缓慢的水域,造成严重的水体富营养化现象,最终导致藻类植物及其它浮游生物过度繁殖。根据富营养化水体中爆发性生长的优势藻的颜色,水面可呈蓝、红、棕等多种颜色。此现象在江河湖泊中被称之为“水华”,而在海洋中则称之为“赤潮”。目前,水体富营养化已成为我国一个较为突出的环境问题。而由有害藻所产生和分泌的次生代谢物——富营养化水体毒素更是因对人类健康的巨大威胁而备受关注。微囊藻毒素(microcystin,MC)是淡水中研究得最为广泛和深入的一种富营养化水体毒素。最初,MC是从铜绿微囊藻(Microcystis aeruginosa)中分离得到的,并因此而被命名为“microcystin"。20世纪80年代初,MC被确认是一种单环七肽类肝毒素。由于其结构的多变性,目前已有80多种微囊藻毒素的异构体被发现,而存在最普遍,毒性最强的则是MCLR。由于MC化学性质稳定,很难将其从水中除去,而该毒素又具有多种毒性效应,因此深入研究其毒性机制有着非常重要的现实意义。大田软海绵酸(Okadaic acid,OA)是多年来海洋赤潮毒素的研究热点,最初从大田软海绵(Halichondria Okadai)和隐瓜海绵(H.melanodocia)中分离得到的。此后,X射线晶体衍射法测得OA的分子结构是一种38碳的聚醚类长链脂肪酸衍生物。作为腹泻性贝毒的主要成分之一,OA引起的腹泻性贝毒中毒虽然已在全球范围内广为报道,但因至今未有OA引起的腹泻而致死的记录,且病情一般在三天后可自行愈合,故该天然毒素的毒性危害并没有在最初得到广泛重视。随着生命科学研究领域的深入发展,OA的多脏器毒性作用已被逐渐发现,有关其毒作用机制的研究正在大量的开展中。在MC和OA的毒性研究中发现,这两种富营养化水体毒素既具有肿瘤促进作用,又具有凋亡诱导能力,而且它们都是蛋白磷酸酶PP2A的强效抑制剂。由于蛋白磷酸化与去磷酸化是真核细胞信号转导的重要调节机制,其动态变化几乎涉及从胚胎发育到个体成熟的所有过程,包括细胞的增殖,分化和凋亡。而蛋白磷酸化与去磷酸化的平衡主要由蛋白激酶(Protein kinases,PK)和蛋白磷酸酶(Protein phosphatases,PPs)调控。因此,对特定蛋白磷酸酶的特异性抑制很可能是造成MC和OA这两种水体毒素的毒作用机制错综复杂的原因之一。然而到目前为止,有关蛋白磷酸酶抑制剂这类富营养化水体毒素与PP2A蛋白表达情况的关联研究尚不多见。而本实验室前期相关研究曾发现微囊藻毒素的另一种异构体MCRR能明显上调人羊膜细胞FL中PP2A-A亚基蛋白的表达。为进一步探索PP2A蛋白表达与毒素类型及毒性效应的相关性,继续研究FL细胞中PP2A-A亚基的蛋白表达在MCLR和OA诱导后的改变情况是有十分重要的意义的。虽然有关MCLR和OA诱导各种细胞凋亡的研究已有大量报道,但这两种富营养化水体毒素的详细毒作用机制至今尚无确切定论。许多实验表明氧化应激和线粒体损伤与MCLR诱导的细胞凋亡有着密切的联系。MCLR的暴露使活性氧ROS的生成增多,继而发生线粒体渗透性转换(mitochondrial permeabilitytransition,MPT),而MPT的发生又导致细胞色素c的释放,并激活下游Caspase的链式反应,最终启动凋亡程序使细胞进入不可逆的死亡状态。但也有研究表明,除经典线粒体途径外,MCLR和OA诱导的细胞凋亡可能存在多种其他的途径。随着凋亡机制的深入研究,内质网应激相关的凋亡途径以及失巢凋亡概念都受到了很大的关注。因此,从多角度研究MCLR和OA诱导的凋亡现象中细胞形态和生化指标的改变,对全面认识这两种富营养化水体毒素的凋亡诱导机制是非常有价值的。在本研究中以MCLR和OA染毒FL细胞,采用MTT法检测细胞活力;用流式细胞仪—PI单染法分析细胞周期的改变情况;用PI/Annexin V双染法及TUNEL法测定毒素诱导的细胞凋亡率;同时通过检测各种凋亡相关因子在染毒细胞内的变化,来探讨各自在细胞凋亡过程中的作用,包括用普通光学倒置显微镜直接观察毒素暴露后细胞形态的变化;用荧光显微镜观察标记了荧光素的鬼笔环肽结合的细胞骨架的排列情况;用荧光标记的Caspase 3抑制剂DEVD-FMK研究胞内凋亡执行酶Caspase 3的活化情况;用Western blot法分析PP2A-A、Chop、Bax、Bcl-2及p53蛋白表达的改变情况,并讨论其与细胞毒性效应的关联;对MCLR染毒后PP2A-A的变化在mRNA水平也做了研究分析;另外,用单细胞凝胶电泳法检测毒素对细胞核DNA的损伤情况。实验结果如下:1.MCLR和OA对FL细胞没有明显的致死毒性,甚至略有促进增殖的作用。2.MCLR对FL细胞周期没有明显影响;OA染毒后可使S期细胞所占比例升高,表明OA处理后使FL细胞受阻于S期。3.MCLR和OA可明显诱导FL细胞发生凋亡,且呈剂量效应关系。4.MCLR对FL细胞的形态和骨架无明显破坏作用;OA染毒后能显著降低细胞的贴壁能力,并使细胞F-actin瓦解、边聚。5.MCLR能明显诱导FL细胞的Caspase 3酶活化,各染毒组与对照组相比升高趋势均有统计学意义,但毒素的暴露浓度与酶活化程度没有对应关系;OA染毒后,各浓度组细胞的Caspase 3酶活化也有不同程度的升高趋势,但中间浓度60 nM和80 nM组的升高趋势不明显。6.MCLR染毒后,FL细胞中PP2A-A、Bax和p53蛋白的表达都呈升高的趋势;Chop蛋白的变化趋势是先升高再下降;而Bcl-2蛋白表达的变化情况则是随着毒素染毒浓度的升高而逐渐下降的趋势。与对照组相比,除Bcl-2外,蛋白的改变大多在低浓度下即出现具有统计学意义的差异,表明MCLR对FL细胞蛋白表达的改变存在灵敏而显著的影响。MCLR对PP2A-A的调节在mRNA水平没有明显表现,各染毒浓度组中PP2A-A mRNA的量与对照组相比无显著差异。7.OA染毒的量效研究表明,PP2A-A和Chop蛋白随染毒浓度的增加呈逐渐升高的趋势,并且自60 nM浓度起与对照组的差异具有统计学意义。而BaX、Bcl-2和p53的蛋白表达情况则有随毒素染毒浓度的升高而逐渐下降的趋势。OA染毒的时效研究表明,BaX、Bcl-2和p53的蛋白表达均随毒素染毒时间的延长呈逐渐下降的趋势。8.MCLR和OA对FL细胞的DNA均有严重的损伤作用。彗星实验中表示DNA损伤程度的两个常用指标尾长和尾相值均与染毒浓度呈正相关,且各浓度组与对照组相比,升高趋势均具有统计学意义。主要结论:1.MCLR染毒后导致细胞DNA的损伤,一方面促进细胞的增殖,另一方面又导致细胞的凋亡。2.MCLR染毒后,响应于细胞DNA的损伤而高表达的p53蛋白通过上调Bax和下调Bcl-2蛋白的表达来共同启动细胞的线粒体凋亡途径。而MCLR暴露后诱导Chop和PP2A-A亚基蛋白的高表达,则提示除线粒体途径外可能尚有其它的凋亡机制存在。3.MCLR染毒导致的Caspase 3的酶活化可能来自经典线粒体途径的激活,也可能是内质网相关的凋亡因子Chop通过对Bel-2的调控而与线粒体途径交汇,最终启动Caspase级联反应而进入凋亡程序。4.OA染毒导致细胞DNA的损伤,既促进细胞的增殖,又导致细胞的凋亡。5.OA对细胞骨架的破坏可能使细胞通过“失巢”的方式来参与凋亡的调节。而凋亡过程中细胞Caspase 3的酶活化程度与染毒浓度无对应关系,表明OA对Caspase 3的活化诱导效应比较复杂,可能有Caspase家族的其他成员参与OA诱导的凋亡机制。6.OA诱导的Chop高表达可能也是凋亡的直接启动因素,通过Bel-2这一膜定位蛋白,将内质网凋亡途径和线粒体凋亡途径紧密连接起来,最终触发多种Caspase级联反应启动凋亡程序。而OA染毒后,p53蛋白表达的变化情况则提示:一方面p53对OA造成的严重DNA损伤无法做出积极反应,引起细胞周期S期的阻滞;另一方面p53对下游Bax无法做出正性调控,需要通过Bel-2家族的其他促凋亡因子来参与凋亡反应。另外,OA在抑制PP2A酶活的同时促进PP2A-A亚基表达的特点使OA的致毒机理更为错综复杂。更多还原

【Abstract】 With the rapid development of civilization, some unscientific human actions in life, industry and agriculture have caused a series of severe environmental problems. A plenty of waste water inpour into numerous stagnant waters including lakes, reservoirs and bays directly, and excessive nutrients (mainly phosphorus and nitrogen) accumulate incessantly in water. All of these lead to the water eutrophication and then result in the outbreak of the algal bloom. Depending upon the characteristic color of predominant species in algal bloom, the water presented different color such as red, blue, brown and so on. The phenomenon occurred in fresh water and marine water was named water bloom and red tide respectively. Since the nature toxins produced by the harmful algal bloom are the potential threats to human health, more and more attention has been paid to their various toxic effects and complicated toxic mechanism.Microcystins (MCs) are the most persistent and widely distributed cyanotoxins produced by freshwater cyanobacteria that belong to the genera Microcystis, Anabaena, Nodularia, Oscillatoria and Nostoc. Among the huge family of cyanobacteria, Microcystis aeruginosa is found to be the most common toxic species. Since 1980s, microcystins had been recognized as a family of monocyclic heptapeptide hepatotoxins. Up to now, more than 80 MC congeners have been found. Microcystin-LR (MCLR) is the most frequently studied as well as the most toxic in the group of MCs. During the recent decades, MCs have received increasing concern worldwide due to their high toxic potential. Okadaic acid (OA) is a marine toxin isolated originally from the sponges Halichondria okadai and H. melanodocia. Subsequently, the structure of OA established by X-ray crystallography showed that the natural product toxin was a polyether fatty acid with a 38 backbone carbon skeleton. Although OA is the major component of diarrhetic shellfish poisoning (DSP) toxins and DSP is widely distributed throughout the world, the toxicological effects of OA was not well studied originally, since clinical symptoms of DSP is usually resolved within 3 days and no human deaths have been reported in literature from cases of DSP. However since OA was found to be widely distributed in the bodies of mice after consumption and was a serious threat to human health, the toxin has gained an increasing interest among researchers because of its complicated biochemical mechanisms.Nowadays it has been well known that MCLR and OA are both potent tumor promoters and they may also act as tumor initiators. Interestingly, they also can induce apoptosis in many cell types. Although the reasons of above-mentioned opposite effects caused by MCLR and OA have not yet been given, it might be explained, at least in part, by their common ability to inhibit protein phosphatases, since a well-balanced network of protein kinases and protein phosphatases is crucial for cells to regulate many important cellular processes, such as proliferation, differentiation and apoptosis. However, to the authors’ knowledge, few studies concerning the relationship between the alteration in protein expression of PP2A and the treatment with the inhibitor of protein phosphatases, such as MCLR and OA, have been published. Interestingly, upregulation of PP2A-A subunit was observed in our most recent study in FL cells treated with microcystin-RR (MCRR), one structural variant among MCs. In order to identify whether or not the upregulation is toxin-specific, the effects of MCLR and OA exposure on protein expression of PP2A-A subunit were studied in the same FL cell model.During recent years, there are numerous studies focused on the apoptosis induced by MCLR and OA. However, the exact mechanisms of their toxicity still have not been fully elucidated. It has been shown that following the MCLR-exposure, the increase of reactive oxidative species (ROS) precedes the onset of mitochondrial permeability transition (MPT) and loss of mitochondrial membrane potential, which lead to cytochrome-c release from the mitochondrion and caspases activation. At last, apoptosis was initiated. All the evidence indicates that mitochondria play a pivotal role in MCLR induced apoptosis. However, in addition to classical mitochondrial pathway, to date, mitochondria-independent pathways of apoptosis induced by many cytotoxin have gained an increasing interest and there has been an emerging view showing that disturbance of cell-anchorage and activation of endoplasmic reticulum stress related components can directly lead to apoptosis. So it was worth paying more attention to all kinds of changes induced by MCLR and OA to provide more evidence for complex apoptosis mechanism.The present study was designed to evaluate the ability of MCLR and OA to induce cytotoxicity. The cell viability was determined by MTT assay. The cell cycle was mensurated by PI staining using flow cytometric analysis. The apoptosis rates induced by MCLR and OA were evaluated by TUNEL assay and PI/Annexin V-FITC double staining respectively. The cell shape change was observed directly by light microscope. The cytoskeleton modification was observed by fluorescent microscope after FITC-phalloidin staining. The Caspase 3 activation was detected by the fluorochrome-labeled inhibitor of Caspase 3. The alteration of protein expression on PP2A-A, Chop, Bax, Bcl-2 and p53 were determined by the western blot. The expression level of PP2A-A mRNA after MCLR exposure was detected by reverse transcription-polymerase chain reaction. Finally the DNA damage was measured by the single cell gel electrophoresis method.The results were shown as below:1. MCLR and OA exposure could induce the proliferation instead of fatal effect in human amnion FL cells.2. There was no obvious impact on cell cycle after MCLR exposure. However the influence of OA to the cell cycle of FL was mainly on the increase of cells that in the S period.3. MCLR and OA exposure induced apoptosis of FL cells in a dose-dependent manner.4. There was no distinct change of cell shape and F-actin distribution after MCLR exposure. However, the FL cells incubated with OA showed loss of cell-to-cell contact, detachment from the substratum and cytoplasmic rounding. In addition, cells showed extensive F-actin depolymerization and focal aggregations of F-actin around the cell periphery.5. MCLR exposure induced the increase of Caspase 3 activation of FL cells and significant differences were observed in all treated groups with respect to control. However, the activation of Caspase 3 induced by OA was significantly increased only in 20 nM, 40 nM and 100 nM OA-treated groups.6. After treatment with MCLR, the expressions of PP2A-A, Bax, and p53 proteins were significantly increased and had statistical significance from the concentration of 10 or 50 nM. In addition, the Chop protein was also upregulated at most treatment groups, although it decreased at the highest concentration group. In contrast, the expression of Bcl-2 seemed to be a concentration-dependent decrease. There were significant differences on its protein levels in 500 and 1000 nM MCLR-treated FL cells compared with control. However, PP2A-A mRNA level of treated groups was not obviously changed compared with that of control group.7. The dose-effect study after OA exposure showed that the levels of PP2A-A and Chop proteins in FL cells were both significantly increased from 60 nM groups. However, the protein expressions of Bax, Bcl-2 and p53 were a dose-dependent decrease. In addition, the time-effect study also showed that the alteration of the three apoptotic-related proteins, Bax, Bcl-2 and p53 were a time-dependent decrease.8. Comet assay showed that both MCLR and OA exposure could induce the DNA damage of FL cells. Both tail length and tail moment increased in a dose-dependent manner.Conclusions:1. MCLR exposure could induce the DNA damage of FL cells. It initiated both proliferation and apoptosis of cells.2. MCLR exposure could increase the expression of p53 protein which is responsible for DNA damage and then triggered apoptosis via a mitochondrial pathway by up-regulation of Bax protein and down-regulation of Bcl-2 protein. The up-regulation of PP2A-A and Chop proteins after MCLR exposure suggested that MCLR might induce apoptosis though more than one pathway.3. The activation of Caspase 3 in MCLR-induced FL cells might be one of the direct events in the mitochondrial pathway of apoptosis process. On the other hand, it also might be due to the indirectly regulation of Chop, one protein involved in the process of apoptosis associated with ER stress, since it has been reported that over-expression of Chop can lead to translocation of Bax protein from the cytosol to the mitochondria and decrease in Bcl-2 protein and finally trigger the mitochondrial pathway.4. OA exposure could induce the DNA damage of FL cells. It initiated both proliferation and apoptosis of cells.5. The change of cell shape and the destruction of F-actin in OA-treated FL cells suggested that OA might trigger a specific form of apoptosis, termed anoikis via the cytoskeletal damage. During the process, the increase of Caspase 3 activation after OA exposure was not in concentration-dependent. It indicated that the response of Caspase 3 activation caused by OA was complicated and there might be multiple caspase isoforms contributed to the apoptosis induced by OA.6. The up-regulation of Chop proteins after OA exposure might play an important role in OA-induced apoptosis, since it has been reported that Chop can directly induce apoptosis as well as can decrease Bcl-2 protein and finally trigger the mitochondrial pathway. And the alteration of p53 protein expression suggested that: the severe DNA damage induced by OA could not been well repaired in time and in effect and many cells were arrested at S phase. Following the change of p53, Bax protein was also decrease, which clued on an anti-apoptotic factor other than Bax might be involved in the apoptosis induced by OA. Finally, the dualistic characteristics of OA, namely it could inhibit PP2A activity as well as increase the expression of PP2A-A might attribute to, at least in part, the complicated apoptosis mechanism induced by OA.更多还原