大田软海绵酸（Okadaic Acid，OA）对小鼠胚胎细胞3T3的影响
摘要
研究背景和研究目的：
在海洋中大约有300种的藻类可以引起赤潮,其中有80种左右可产生毒素。这些毒素可以通过鱼类、贝类等海洋生物的富集传递作用引起人类中毒,对人类健康形成了潜在威胁。因此许多国家都建立了赤潮检测体系,并对赤潮藻毒素的毒性作用机制及检测方法做了一系列的研究,取得了重大进展。
然而,迄今为止,对于利马原甲藻等有害赤潮藻所产生等藻毒素对于小鼠细胞的细胞内部结构、增殖和凋亡的的影响的研究还很少,且对于对有害藻类的研究主要集中在淡水蓝藻,其余藻类涉及的较少。因此,我们有必要对有害赤潮藻对于哺乳动物细胞的影响进行全面的研究。为在细胞水平上提供对赤潮藻毒素毒性的检测以及对赤潮的危害的预报上提供更为准确的数据。
研究方法：
1.MTT及细胞计数法检测细胞存活率,检测不同浓度OA对细胞数量的影响,对OA毒性强度进行初步确定。
2.细胞凋亡情况的检测：
—倒置相差显微镜观察细胞形态变化；
—吖啶橙(AO)荧光染色,结合激光共聚焦显微镜观察进一步检测细胞凋亡形态变化；
—活性氧(ROS)含量测定,用荧光分光光度计检测细胞内活性氧的含量；
—中性红染色,倒置显微镜观察细胞中性红的摄入量以检测细胞凋亡中细胞膜活性变化；
—乳酸脱氢酶(LDH)含量检测,用乳酸脱氢酶试剂盒结合紫外分光光度计检测细胞内LDH含量的变化；
3.细胞周期影响的检测：
—流式细胞技术检测OA对细胞周期的影响。
4.OA对caspase-3激活情况的检测：用caspase-3活性检测试剂盒分析细胞内caspase-3酶活性变化；
研究结果：
1.OA及利马原甲藻提取物对小鼠胚胎细胞3T3细胞增殖的影响
结果显示：当藻浓度在0.2×103cells//ml时,藻类提取液对细胞抑制较弱。随着藻浓度增大,藻类提取液对细胞增殖的抑制程度逐渐增加,且与时间呈正相关。OA可以显著抑制3T3细胞的生长,且对3T3细胞的增殖抑制效应呈现出明显的剂量依赖性,即随药物浓度的提高,细胞存活率逐渐下降。
2.OA对3T3细胞的内外部形态影响—倒置相差显微镜观察细胞形态变化结果表明：经10ng/mLOA处理后,细胞部分变圆,边缘皱缩并有细胞膜发泡现象；暴露于50ng/mL的细胞绝大部分失去梭形形状,细胞相互分离边缘,可以见到由细胞膜包裹的凋亡小体；而暴露于100ng/mL的细胞凋亡小体明显可见并出现不同程度的破裂
—吖啶橙(AO)染色结合荧光显微镜检测染色质凝集结果
结果显示：OA作用后,细胞染色增强,荧光更为明亮,染色质呈固缩状或圆珠状(早期凋亡)；随着OA浓度增加,细胞和染色质呈固缩状或圆珠状,呈典型的细胞凋亡的特征
3. OA对3T3细胞膜透性的影响
—中性红染色实验
结果显示：50ng/ml和100ng/ml的OA分别处理3T3细胞48小时,与对照组相比,实验组细胞摄入中性红的量明显增加,并伴有细胞体积变小固缩、胞质内空泡化明显。这说明,随着OA浓度增加,细胞膜的通透性增加。
—LDH检测细胞膜完整性
结果显示：50ng/ml和100ng/ml的OA分别处理3T3细胞48小时,与对照组相比,实验组培养液中LDH酶活性与对照组相比变化显著(p<0.05,n=3)。这表明,高浓度处理组中细胞出现了坏死的现象。
4. OA对3T3细胞凋亡的影响
一细胞内活性氧水平的检测
结果显示：检测结果显示：3T3细胞经OA处理48小时,引起3T3细胞内ROS得大量产生和聚集。与OA对照组相比,50ng/mlOA处理组ROS的水平从4.408升高至9.079,增加了2.05倍,而经过100ng/mlOA处理后,ROS水平再次上升为22.6,增长了5.12倍。
—Caspase-3酶活性的检测
结果显示：Caspase-3酶活检测试剂盒检测分析发现：50ng/ml的OA处理3T3细胞48小时后,与对照组相比,405nm处光吸收值由0.0425上升至0.133,活性上调了2.9倍；
—流式细胞仪检测细胞周期分布结果显示：OA可诱导3T3细胞凋亡并将其细胞周期阻滞于G1期使细胞不能进入S期。
Background and objective:
In the oceans there are approximately 300 species of algae that can cause red tides, among which there are about 80 species can produce toxins. These toxins can poison and form a potential threat human through the fish, shellfish of marine life through enrichment transfer function. So governments attach great importance to the harm of red tide phycotoxins, and many countries have established red tides testing system.They made a series of research to the toxins toxicity mechanism and detection method of red tide phycotoxins which has made significant progress.
However, so far, the studys of the impact of Prorocentrum lima Dorge to the ultrastructure, proliferation and apoptosis of mice cells are rare, and the studys mainly focus on freshwater algae, the remaining algae involving less. Therefore, it is necessary for us to have comprehensive research of the influence of harmful algal red tides to mamm alian cells, for providing accurate data on the detection of he toxicity of red tide phycotoxins and the prediction of red tide on cellular level.
Methods:
1. MTT assay on cell viability, by which the toxicity strength of OA was determined primarily.
2. The cell count, detection of influence of different concentrations on OA cell count.
3. Detection of cell apoptosis
—Observation of cell morphological changes by Phase Contrast Microscope.
—Observation of morphological changes of apoptosis by Acridine orange (AO) fluorescence staining combined with Laser Confocal Scanning Microscopy.
—Detection of Reactive oxygen species (ROS), with fluorescence spectrophotome- ter detection intracellular ROS content.
—Detection of the neutral red stain, observing cells to detect the intake of neutral red of cell apoptosis of the activity cell membranes changes with Inverted Microscopy.
—Detection of Lactate dehydrogenase (LDH), detection intracellular LDH content change with lactate dehydrogenase kit combined with UV spectrophotometer.
4. Detection of changes in cell cycle distribution
—Flow cytometry was used to detect the changes about the cell cycle of 3T3 after treated with OA
5. Detection of the activity of OA to caspase-3:analysis the activity change of caspase-3 enzyme intracellular with caspase-3 active immuosorbent.
Results:
1. The test results of MTT assay on cells livability
—Influences of the extract of P. lima on viability of 3T3 cell
When algae concentration in 0.2×103cells/ml, the extract of algae inhibit weakly to cellular. With algae concentration increases, the reduction of extract of algae on cell proliferation is gradually increasing, and positively correlated with time. OA can significantly inhibit 3T3 cell growth and presented dose dependent on 3T3 cell proliferation inhibiting effect obviously. Namely with the increase of the concentration of the drug, cell survival rate fall gradually.
—OA can significantly inhibit 3T3 cell growth, and the proliferation inhibiting effect obviously presented dose dependent on 3T3 cell Namely with the increase of the concentration of the drug, cell survival rate fall gradually.
2. The influence of OA on the internal and external form of 3T3 cell
—Observing cells form changes with inverted differ microscope
Results show that:cells appear part and go round in 10 ng/mL, edge knits and cell membrane foaming phenomenon; Exposure in 50 ng/mL, most cells lose their spindle shape, the cells apart from each edge.We can see that a membrane-bound parcel apoptotic body forms. And exposure to 100 ng/mL, cells apoptotic body is visible and the emergence of different degrees of rupture.
The results of condensed chromatin with Acridone acetamiprid orange (AO) dyeing combined with laser confocal microscopy
The result shows:after the OA dyeing, the cells dyeing enhancement, the fluorescence became more bright, the euchromatin became pyknotic form or round shape(early apoptosis); With the concentration increasing, cells and euchromatin is become pyknotic form or chromatin round shape, a typical cell apoptosis characteristics appears.
3. The influence of OA on 3T3 cell membrane permeability
—The neutral red dyeing experiments
The result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, the experimental group neutral red cell intake volume increased obviously, and accompanied by cells go round. This shows that with increasing concentration, membrane permeability increase.
—Detection of membrane integrity with LDH kit
The result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, in the medium of experimental group, LDH enzyme activity changes significantly (p<0.05, n=3). This suggests that high density dealing group of cells appeared necrosis phenomenon.
4.The influence of OA on the apoptosis of 3T3 cell
—Detection of intracellular ROS levels
The result shows:after OA process 48 hours, the ROS in 3T3 cell erupt and gathered. Compared with control group, levels of ROS in 50ng/ml OA treatment groups from 4.408 elevated to 9.079, increased 2.05 times, and after 100ng/ml OA processing, ROS levels rise again to 22.6, increased 5.12 times as many.
—Detection of Caspase-3 enzyme activity
The result shows:the Caspase-3 enzymes living immuosorbent detection analysis shows that, 50ng/ml of OA processing 3T3 cell for 48 hours, compared with control group, light absorption value in 405 nm 0.0425 rose to 0.133, active raised 2.9 times.
—The result shows:OA can induce 3T3 cell apoptosis and blockade thecell cycle in G1 phase make cells can't enter S period.
在海洋中大约有300种的藻类可以引起赤潮,其中有80种左右可产生毒素。这些毒素可以通过鱼类、贝类等海洋生物的富集传递作用引起人类中毒,对人类健康形成了潜在威胁。因此许多国家都建立了赤潮检测体系,并对赤潮藻毒素的毒性作用机制及检测方法做了一系列的研究,取得了重大进展。
然而,迄今为止,对于利马原甲藻等有害赤潮藻所产生等藻毒素对于小鼠细胞的细胞内部结构、增殖和凋亡的的影响的研究还很少,且对于对有害藻类的研究主要集中在淡水蓝藻,其余藻类涉及的较少。因此,我们有必要对有害赤潮藻对于哺乳动物细胞的影响进行全面的研究。为在细胞水平上提供对赤潮藻毒素毒性的检测以及对赤潮的危害的预报上提供更为准确的数据。
研究方法：
1.MTT及细胞计数法检测细胞存活率,检测不同浓度OA对细胞数量的影响,对OA毒性强度进行初步确定。
2.细胞凋亡情况的检测：
—倒置相差显微镜观察细胞形态变化；
—吖啶橙(AO)荧光染色,结合激光共聚焦显微镜观察进一步检测细胞凋亡形态变化；
—活性氧(ROS)含量测定,用荧光分光光度计检测细胞内活性氧的含量；
—中性红染色,倒置显微镜观察细胞中性红的摄入量以检测细胞凋亡中细胞膜活性变化；
—乳酸脱氢酶(LDH)含量检测,用乳酸脱氢酶试剂盒结合紫外分光光度计检测细胞内LDH含量的变化；
3.细胞周期影响的检测：
—流式细胞技术检测OA对细胞周期的影响。
4.OA对caspase-3激活情况的检测：用caspase-3活性检测试剂盒分析细胞内caspase-3酶活性变化；
研究结果：
1.OA及利马原甲藻提取物对小鼠胚胎细胞3T3细胞增殖的影响
结果显示：当藻浓度在0.2×103cells//ml时,藻类提取液对细胞抑制较弱。随着藻浓度增大,藻类提取液对细胞增殖的抑制程度逐渐增加,且与时间呈正相关。OA可以显著抑制3T3细胞的生长,且对3T3细胞的增殖抑制效应呈现出明显的剂量依赖性,即随药物浓度的提高,细胞存活率逐渐下降。
2.OA对3T3细胞的内外部形态影响—倒置相差显微镜观察细胞形态变化结果表明：经10ng/mLOA处理后,细胞部分变圆,边缘皱缩并有细胞膜发泡现象；暴露于50ng/mL的细胞绝大部分失去梭形形状,细胞相互分离边缘,可以见到由细胞膜包裹的凋亡小体；而暴露于100ng/mL的细胞凋亡小体明显可见并出现不同程度的破裂
—吖啶橙(AO)染色结合荧光显微镜检测染色质凝集结果
结果显示：OA作用后,细胞染色增强,荧光更为明亮,染色质呈固缩状或圆珠状(早期凋亡)；随着OA浓度增加,细胞和染色质呈固缩状或圆珠状,呈典型的细胞凋亡的特征
3. OA对3T3细胞膜透性的影响
—中性红染色实验
结果显示：50ng/ml和100ng/ml的OA分别处理3T3细胞48小时,与对照组相比,实验组细胞摄入中性红的量明显增加,并伴有细胞体积变小固缩、胞质内空泡化明显。这说明,随着OA浓度增加,细胞膜的通透性增加。
—LDH检测细胞膜完整性
结果显示：50ng/ml和100ng/ml的OA分别处理3T3细胞48小时,与对照组相比,实验组培养液中LDH酶活性与对照组相比变化显著(p<0.05,n=3)。这表明,高浓度处理组中细胞出现了坏死的现象。
4. OA对3T3细胞凋亡的影响
一细胞内活性氧水平的检测
结果显示：检测结果显示：3T3细胞经OA处理48小时,引起3T3细胞内ROS得大量产生和聚集。与OA对照组相比,50ng/mlOA处理组ROS的水平从4.408升高至9.079,增加了2.05倍,而经过100ng/mlOA处理后,ROS水平再次上升为22.6,增长了5.12倍。
—Caspase-3酶活性的检测
结果显示：Caspase-3酶活检测试剂盒检测分析发现：50ng/ml的OA处理3T3细胞48小时后,与对照组相比,405nm处光吸收值由0.0425上升至0.133,活性上调了2.9倍；
—流式细胞仪检测细胞周期分布结果显示：OA可诱导3T3细胞凋亡并将其细胞周期阻滞于G1期使细胞不能进入S期。
Background and objective:
In the oceans there are approximately 300 species of algae that can cause red tides, among which there are about 80 species can produce toxins. These toxins can poison and form a potential threat human through the fish, shellfish of marine life through enrichment transfer function. So governments attach great importance to the harm of red tide phycotoxins, and many countries have established red tides testing system.They made a series of research to the toxins toxicity mechanism and detection method of red tide phycotoxins which has made significant progress.
However, so far, the studys of the impact of Prorocentrum lima Dorge to the ultrastructure, proliferation and apoptosis of mice cells are rare, and the studys mainly focus on freshwater algae, the remaining algae involving less. Therefore, it is necessary for us to have comprehensive research of the influence of harmful algal red tides to mamm alian cells, for providing accurate data on the detection of he toxicity of red tide phycotoxins and the prediction of red tide on cellular level.
Methods:
1. MTT assay on cell viability, by which the toxicity strength of OA was determined primarily.
2. The cell count, detection of influence of different concentrations on OA cell count.
3. Detection of cell apoptosis
—Observation of cell morphological changes by Phase Contrast Microscope.
—Observation of morphological changes of apoptosis by Acridine orange (AO) fluorescence staining combined with Laser Confocal Scanning Microscopy.
—Detection of Reactive oxygen species (ROS), with fluorescence spectrophotome- ter detection intracellular ROS content.
—Detection of the neutral red stain, observing cells to detect the intake of neutral red of cell apoptosis of the activity cell membranes changes with Inverted Microscopy.
—Detection of Lactate dehydrogenase (LDH), detection intracellular LDH content change with lactate dehydrogenase kit combined with UV spectrophotometer.
4. Detection of changes in cell cycle distribution
—Flow cytometry was used to detect the changes about the cell cycle of 3T3 after treated with OA
5. Detection of the activity of OA to caspase-3:analysis the activity change of caspase-3 enzyme intracellular with caspase-3 active immuosorbent.
Results:
1. The test results of MTT assay on cells livability
—Influences of the extract of P. lima on viability of 3T3 cell
When algae concentration in 0.2×103cells/ml, the extract of algae inhibit weakly to cellular. With algae concentration increases, the reduction of extract of algae on cell proliferation is gradually increasing, and positively correlated with time. OA can significantly inhibit 3T3 cell growth and presented dose dependent on 3T3 cell proliferation inhibiting effect obviously. Namely with the increase of the concentration of the drug, cell survival rate fall gradually.
—OA can significantly inhibit 3T3 cell growth, and the proliferation inhibiting effect obviously presented dose dependent on 3T3 cell Namely with the increase of the concentration of the drug, cell survival rate fall gradually.
2. The influence of OA on the internal and external form of 3T3 cell
—Observing cells form changes with inverted differ microscope
Results show that:cells appear part and go round in 10 ng/mL, edge knits and cell membrane foaming phenomenon; Exposure in 50 ng/mL, most cells lose their spindle shape, the cells apart from each edge.We can see that a membrane-bound parcel apoptotic body forms. And exposure to 100 ng/mL, cells apoptotic body is visible and the emergence of different degrees of rupture.
The results of condensed chromatin with Acridone acetamiprid orange (AO) dyeing combined with laser confocal microscopy
The result shows:after the OA dyeing, the cells dyeing enhancement, the fluorescence became more bright, the euchromatin became pyknotic form or round shape(early apoptosis); With the concentration increasing, cells and euchromatin is become pyknotic form or chromatin round shape, a typical cell apoptosis characteristics appears.
3. The influence of OA on 3T3 cell membrane permeability
—The neutral red dyeing experiments
The result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, the experimental group neutral red cell intake volume increased obviously, and accompanied by cells go round. This shows that with increasing concentration, membrane permeability increase.
—Detection of membrane integrity with LDH kit
The result shows:OA act on the 3T3 cell 50ng/ml and 100ng/ml respectively processing 48 hours, compared with control group, in the medium of experimental group, LDH enzyme activity changes significantly (p<0.05, n=3). This suggests that high density dealing group of cells appeared necrosis phenomenon.
4.The influence of OA on the apoptosis of 3T3 cell
—Detection of intracellular ROS levels
The result shows:after OA process 48 hours, the ROS in 3T3 cell erupt and gathered. Compared with control group, levels of ROS in 50ng/ml OA treatment groups from 4.408 elevated to 9.079, increased 2.05 times, and after 100ng/ml OA processing, ROS levels rise again to 22.6, increased 5.12 times as many.
—Detection of Caspase-3 enzyme activity
The result shows:the Caspase-3 enzymes living immuosorbent detection analysis shows that, 50ng/ml of OA processing 3T3 cell for 48 hours, compared with control group, light absorption value in 405 nm 0.0425 rose to 0.133, active raised 2.9 times.
—The result shows:OA can induce 3T3 cell apoptosis and blockade thecell cycle in G1 phase make cells can't enter S period.
引文
1.陆斗定.全球赤潮生态学与海洋学(GEOHAB),国际合作计划[J].东海海洋.2002,20(1)：60-64.
2. Kevin G. Sellner Gregory J. Doucette Gary J. Kirkpatrick. Harmful algal blooms:causes, impacts and detection [J]. Ind Microbiol Biotechnol.2003,30:383-406.
3. Adams NG, Lesoing M, Trainer VL, Environmental conditions associated with domoic acid in razor clams on the Washington coast [J]. Shellfish Research.2000,19:1007-1015.
4. Folke C, Kautsky N, Troell M. Salmon farming in context:response to Black et al [J]. Environ Manage.1997,50:95-103.
5. Rensel J E, Whyte J C. Finfish mariculture and harmful algal blooms. In:Hallegraeff GM, Anderson DM, Cembella AD (eds) Manual on harmful marine microalgae [J]. Monographs on oceanographic methodology.2003,11(25):55-59.
6. Smayda T J. Novel and nuisance phytoplankton blooms in the sea-evidence for a global epidemic [J]. Toxic marine phytoplankton.1990,12:29-40.
7. Okaichi T. Red tide problems in the Seto Inland Sea [J]. Environmental Science and Toxicology.1989,11:137-144.
8. Tasumoto T, Oshima M Y, Yamaguchi M. Occurrence of a new type of shellfish poisoning in the Tohoku District [J]. Bull Jpn Soc Sci Fish.1978,44:1249-1255.
9.杜伟,陆斗定.有毒赤潮藻及其毒素的危害与检测[J].海洋学研究.2008,26(2)：89-96.
10.卢士英,柳增善等.海产品贝毒素大田软海绵酸ELISA及HPLC-MS/MS检测方法的研究[J].食品科学分析.2009,30(08)：158-162.
11.秦路平.生物活性成分的高通量筛选[M].上海：第二军医大学出版社,2001.
12. Cohen P, Holmes C F, Tsukitani T. Okadaic acid:a new probe forthe study of cellular regulation [J]. TBIS.1990,15:98-102.
13. Viviani R. Eutrophication, marinebiotoxins, human health [J]. Science of the Total Environment.1992,38:631-633.
14.敖琳,高利宏等.岗田酸促癌作用过程中对Balb/c 3T3细胞凋亡的影响[J].中国药理学与毒理学杂志.2006,20(5)：414-420.
15. Green D R, Rees J C. Mitochondrion and apoptosis [J]. Science.1998, (281):1309-1312.
16. Jacobson M D, Weil M, Raff M C.Programmed cell death in animal development [J]. Cell. 1997,88:347-365.
17. Ashkenazi A, Dixit V M. Death receptors:signaling and modulation [J]. Science.1998, 281(53):1305-1308.
18. Pan G, O'Rourke K, Chinnaiyan A M, et al. The receptor for the cytotoxic ligand TRAIL [J]. Science.1997,276(53):111-113.
19. Screaton G R, Mongkolspaya J, Xu X N et al. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL [J]. Curr Biol.1997,7(9):693-696.
20.任满意,张运,隋树建.肿瘤坏死因子相关的凋亡诱导配体及其死亡受体与急性冠状动脉综合征的关系[J].中国动脉硬化杂志.2007,15(11)：842-846.
21. Villa Morales M, Santos J, Perez Gomez E, et al. A role for the Fas/FasL system in modulating genetic susceptibility to T-cell lymphoblastic lymphomas [J]. Cancer Res.2007,67(11)：5107-5116.
22.陈儒新,尹福波.死亡受体、配体系统与卵巢肿瘤[J].国外医学计划生育分册.2005,24(4)：208-212.
23. Schulze Osthoff K, Ferrari D, LosM, et al. Structural and biochemical basis of TNS-3 cell [J]. Eur J Biochem.1998,254(3):439-459.
24. Creagh E M, Conroy H, Martin SJ. Caspase activation pathways in apoptosis and immunity [J]. Immunol Rev.2003,193:10-21.
25. Li H, Zhu H, Xu C J. Cleavafe of BID by caspase-8 mediates mitochondrial damage in the Fas pathway of apoptosis [J]. Cell.1998,94(4);491-501.
26. Eberstadt M, Huang B, Chen Z, et al. NMR structure and mutagenesis of the FADD (Mortl) death-effector domain [J]. Nature.1998,392(9):941-945.
27. Adrain C, Martin S J. The mitochondrial apoptosome:a killer unleashed by the cytochrome seas [J]. Trends Biochem Sci.2001,26(6):390-397.
28. Webster C R, Usechiak P, Anwer M S. cAMP inhibits bile acid induced apoptosis by blocking caspase activation and cytochrome-c release [J]. Cell.2002,283:G727-G738.
29.郑训针,张广森.线粒体介导的凋亡途径及调控机制[J].中华血液学杂志.2001,22(1)：52-54.
30. Rupniewska Z, Bojarskajuna. Apoptosis:mitochondrial membrane permesbili-zation and the role played by Bcl-2 family proteins [J]. Cell.2004.58:538-547.
31. Chas J, Du C. Structural and biochemical basis of apoptotic activation by Smac/DIABLO [J]. Nature.2000(406):855-862.
32. Yoshide B, Kang Y Y, Yoshide R, et al. Apaf-1 is required form itochondrial pathways of apoptosis and brain development [J]. Cell.1998,94:739-750.
33. Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxi collett.2004,149(1-3)：19-23.
34. Szegezdi, Fitzgerald U, Samali A. Caspase-12 and ER-stress-mediated apoptosis:the story so far [J]. Ann NY Acid Sci.2003,1010:186-194.
35. Ferri K F, Kroemer G. Organelle specific initiation of cell death pathways [J]. Nat Cell Biol.2001,31(11):255-263.
36.姜山,谢青.内质网应激与细胞凋亡[J].国外医学流行病传染病学分册.2004,31(6)：330-333.
37. Li H, Yuan J Y. Decophering the pathways of life and death [J]. Curr Op in Cell Bio.1999, 11:261-266.
38. Lang R. Rapid colorimetric assay for cell growth and surrvial:Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability [J]. J Immunol Methods.1986,89(2):271-277.
39. Twentyman P R, Luscombe M. A study of some variables in a tetrazolium dye (MTT) bassed assay for cell growth and chemosensitxity [J]. BRJ Cancer.1987,56(3):279-285.
40. Traore A, Baudrimont I. Ambaliou S, et al. DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line [J]. Arch Toxico.2001,75(2)：110-117.
41. Lin XY, Choi MS, Porter AG. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma [J]. Biol Chem.2000,275(51):920-926.
42. Rao RV, Peel A, Logvinova A, et al. Coupling endoplasmic reticulum stress to the cell death program:role of the ER chaperone GRP7 [J]. FEBS Lett.2002(2),514:122-128.
43.黄爱君,黄海燕,庄志雄等.大田软海绵酸对HL-7702肝细胞毒性效应的研究[J].安徽农业科学.2010,38(10)：521-525.
44. Vera Ritz, John Marwitz, Sabine S. Iris Quentin. Contribution of mdrlb-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells [J]. Naunyn Schmiedeberg's Arch Pharmacol.1999,360:116-121.
45. Kerr J F, Wyllie A H. Apoptosis:a basic biological phenomenon with wide ranging locations in tissue kenetics [J]. Journal of Cancer.1972,26:239-245.
46. Creagh E M, Conroy H, Martin S J. Caspase activation pathways in apoptosis and immunity [J]. Immunol Rev.2003,193:10-21.
47. Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase-8 mediates mitochondrial damage in the Fas pathway of apoptosis [J]. Cell.1998,94(4):491-501.
48. Adrain C, Martin S J. The mitochondrial apoptosome:a killer unleashed by the cytochrome seas [J]. Trends Biochem Sci.2001,26(6):390-397.
49.陈洋.DSP等赤潮藻毒素对哺乳类细胞的毒性效应及研究机制[博士学位论文].中国海洋大学：周名江,2008年.
50. Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxi collett.2004, 149(1-3)：19-23.
51. Kimura K, Sasano H, Shimosegawa T, et al. Ultrastructure of cells undergoing apoptosis [J]. Vitam Horm.2000,58:257-266.
52. Hacker G. The morphology of apoptosis [J]. Cell Tissue Res.2000,301(1):5-17.
53. Trump BF, Berezesky IK, Chang SH, et al. The pathways of cell death:oncosis, apoptosis, and necrosis [J]. Toxicol Pathol.1997,25(1):82-86.
54.李沂轩,纪岩文：吖啶橙染色方法检查线索细胞[J].中国皮肤性病学杂志.2005,19(11)：697.
55.廖二元,杨川,戴如春.激光共聚焦显微镜在骨计量学中的应用[J].湖南医科大学学报,2000,25(4)：413-415.
56. Chiba K, Kawakami K, Tohyama K. Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells [J]. Toxicol In Vitro.1998,12:251-258.
57. Steinman RM, Mellman IS, Muller WA, et al. Endocytosis and the recycling of plasma membrane [J]. Cell Biol.1983,96(1):1-27.
58.李振荣,张捷,王天成等.对乳酸脱氢酶同工酶谱的异常区带和增高的乳酸脱氢酶1组织来源的分析[J].北京大学学报(医学版).2004,12(5)：552-553.
59.刘锐克,周军兰,梁建辉.中性红比色法检测大鼠小脑神经瘤活细胞[J].中国药理学与毒理学杂志.2003,17(1)：74-78.
60.孙皎,高桑美代子,佐藤和子等.中性红染色法定量检测牙科材料细胞毒性的研究[J].中国生物医学工程学报.1999,18(1)：118-120.
61. Schwartzman R, Cidlowski J. Apoptosis:the biochemistry and molecular biology of programmed cell death [J]. Endocrine Rev.1993,14:133.
62. Vermes, I. and Haanan, C. Apoptosis and programmed cell death in health and disease [J]. Adv. Clin. Chem.1994,31,177.
63. Majno G, Joris I. Apoptosis, oncosis, and necrosis:An overview of cell death [J]. Am J Pathol.1995,146(1):3-15.
64. Zimmermann K C, Bonzon C, Green D R. The machinery of programmed cell death [J]. Pharmacol Ther.2001,92(1):57-70.
65. Neiman P E, Thomas S J, Loring G. Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius [J]. Proc Natl Acad Sci U S A.1991,55:5857-5861.
66. Berke G. Debate:the mechanism of lymphocyte-mediated killing Lymphocyte triggered internal target disintegration [J]. Innunol Today.1991,12:396.
67. Suppression of tumo necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control [J]. Proc Natl cad Sci.1997,56:57-62.
68. Khoshnan A, Tindell C. The NF-kappa Bcascade is important in Bclxl expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4 lymphocytes [J]. Immunol.2000,165:1743-1754.
69. Stehlik C, Martin R, Kumabashiri, et al. Nuclear factor(NF)-kappa-B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis [J]. Exp Med.1998,58:211-216.
70. Asso-Bonnet M, Feuillard J. Relationship between kappa B alpha constitutive expression, TNF alpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells [J]. Oncogene.1998,59(17)：1607-1615.
71. Lee K Y, Chang W, Qiu D, et al. PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells [J]. Biol Chem.1999,60:451-455.
72. Sugiyama H, Savill J S. Selective sensitization to tumor necrosis factor alpha induced apoptosis by blockade of NF kappa B in primary glomerular mesangial cells [J] Biol Chem.199961：532-537.
73. Shao R, Karunagaran D, Zhou B. P, et al. Inhibition of nuclear factor-kappa B activity is involved in ElA-mediated sensitization of radiation-inducedapoptosis [J]. Biol Chem.1997,62：739-742.
74. Kajino S, Suganuma M, Teranishi F et al. Evidence that de novo protein synthesis is dispensable for anti-apoptotic-effects of NF-kappaB [J]. Oncoaene.2000,63:233-239.
75. Javelaud D, Besancon F. NF-kappa B activation results in rapid inactivation of JNK in TNF alpha treated Ewing sarcoma cells:a mechanism for the anti-apoptotic effect of NF-kappa B [J]. Oncoaene.200164:365-372.
76. Martindale J L, Holbrook N J. Cellular response to oxidative strees:signaling for suicide and survival [J]. Cell Physio.2002,192(1):1-15.
77. Dypbukt J M, Ankarcrona M, Burkitt M et al. Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines [J]. Biol Chem.1994,269(48):553-560.
78. Lin, X Y, Choi M S, Porter A G. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma [J]. Biol Chem.2000,275:920-926.
79. Vinella D, D'Ari R. Overview of controls in the Escherichia coli cell cycle [J]. Bioessays.1995,17(6)：527-536.
80. Norbury C, Nurse P. Animal cell cycles and their control [J]. Ann Rev Biochem.1992,61: 441-470.
81. Bergrnann M. W, Loser P, Dietz R, et al. Effect of NF kappa B Inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes [J]. Mol Cell Cardiol.2001,65:1223-1232.
82. Klumpp S, Rieglstein J. Serine/threonine protein phosphatases in apoptosis [J]. Curr Opin Pharmaco.2002,2(4):458-462.
83. Liu J P, Schmid P C. Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 tomodulate its transcriptional and apoptotic activities [J]. Oncogene. 2006,25(21):3006-3022.
2. Kevin G. Sellner Gregory J. Doucette Gary J. Kirkpatrick. Harmful algal blooms:causes, impacts and detection [J]. Ind Microbiol Biotechnol.2003,30:383-406.
3. Adams NG, Lesoing M, Trainer VL, Environmental conditions associated with domoic acid in razor clams on the Washington coast [J]. Shellfish Research.2000,19:1007-1015.
4. Folke C, Kautsky N, Troell M. Salmon farming in context:response to Black et al [J]. Environ Manage.1997,50:95-103.
5. Rensel J E, Whyte J C. Finfish mariculture and harmful algal blooms. In:Hallegraeff GM, Anderson DM, Cembella AD (eds) Manual on harmful marine microalgae [J]. Monographs on oceanographic methodology.2003,11(25):55-59.
6. Smayda T J. Novel and nuisance phytoplankton blooms in the sea-evidence for a global epidemic [J]. Toxic marine phytoplankton.1990,12:29-40.
7. Okaichi T. Red tide problems in the Seto Inland Sea [J]. Environmental Science and Toxicology.1989,11:137-144.
8. Tasumoto T, Oshima M Y, Yamaguchi M. Occurrence of a new type of shellfish poisoning in the Tohoku District [J]. Bull Jpn Soc Sci Fish.1978,44:1249-1255.
9.杜伟,陆斗定.有毒赤潮藻及其毒素的危害与检测[J].海洋学研究.2008,26(2)：89-96.
10.卢士英,柳增善等.海产品贝毒素大田软海绵酸ELISA及HPLC-MS/MS检测方法的研究[J].食品科学分析.2009,30(08)：158-162.
11.秦路平.生物活性成分的高通量筛选[M].上海：第二军医大学出版社,2001.
12. Cohen P, Holmes C F, Tsukitani T. Okadaic acid:a new probe forthe study of cellular regulation [J]. TBIS.1990,15:98-102.
13. Viviani R. Eutrophication, marinebiotoxins, human health [J]. Science of the Total Environment.1992,38:631-633.
14.敖琳,高利宏等.岗田酸促癌作用过程中对Balb/c 3T3细胞凋亡的影响[J].中国药理学与毒理学杂志.2006,20(5)：414-420.
15. Green D R, Rees J C. Mitochondrion and apoptosis [J]. Science.1998, (281):1309-1312.
16. Jacobson M D, Weil M, Raff M C.Programmed cell death in animal development [J]. Cell. 1997,88:347-365.
17. Ashkenazi A, Dixit V M. Death receptors:signaling and modulation [J]. Science.1998, 281(53):1305-1308.
18. Pan G, O'Rourke K, Chinnaiyan A M, et al. The receptor for the cytotoxic ligand TRAIL [J]. Science.1997,276(53):111-113.
19. Screaton G R, Mongkolspaya J, Xu X N et al. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL [J]. Curr Biol.1997,7(9):693-696.
20.任满意,张运,隋树建.肿瘤坏死因子相关的凋亡诱导配体及其死亡受体与急性冠状动脉综合征的关系[J].中国动脉硬化杂志.2007,15(11)：842-846.
21. Villa Morales M, Santos J, Perez Gomez E, et al. A role for the Fas/FasL system in modulating genetic susceptibility to T-cell lymphoblastic lymphomas [J]. Cancer Res.2007,67(11)：5107-5116.
22.陈儒新,尹福波.死亡受体、配体系统与卵巢肿瘤[J].国外医学计划生育分册.2005,24(4)：208-212.
23. Schulze Osthoff K, Ferrari D, LosM, et al. Structural and biochemical basis of TNS-3 cell [J]. Eur J Biochem.1998,254(3):439-459.
24. Creagh E M, Conroy H, Martin SJ. Caspase activation pathways in apoptosis and immunity [J]. Immunol Rev.2003,193:10-21.
25. Li H, Zhu H, Xu C J. Cleavafe of BID by caspase-8 mediates mitochondrial damage in the Fas pathway of apoptosis [J]. Cell.1998,94(4);491-501.
26. Eberstadt M, Huang B, Chen Z, et al. NMR structure and mutagenesis of the FADD (Mortl) death-effector domain [J]. Nature.1998,392(9):941-945.
27. Adrain C, Martin S J. The mitochondrial apoptosome:a killer unleashed by the cytochrome seas [J]. Trends Biochem Sci.2001,26(6):390-397.
28. Webster C R, Usechiak P, Anwer M S. cAMP inhibits bile acid induced apoptosis by blocking caspase activation and cytochrome-c release [J]. Cell.2002,283:G727-G738.
29.郑训针,张广森.线粒体介导的凋亡途径及调控机制[J].中华血液学杂志.2001,22(1)：52-54.
30. Rupniewska Z, Bojarskajuna. Apoptosis:mitochondrial membrane permesbili-zation and the role played by Bcl-2 family proteins [J]. Cell.2004.58:538-547.
31. Chas J, Du C. Structural and biochemical basis of apoptotic activation by Smac/DIABLO [J]. Nature.2000(406):855-862.
32. Yoshide B, Kang Y Y, Yoshide R, et al. Apaf-1 is required form itochondrial pathways of apoptosis and brain development [J]. Cell.1998,94:739-750.
33. Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxi collett.2004,149(1-3)：19-23.
34. Szegezdi, Fitzgerald U, Samali A. Caspase-12 and ER-stress-mediated apoptosis:the story so far [J]. Ann NY Acid Sci.2003,1010:186-194.
35. Ferri K F, Kroemer G. Organelle specific initiation of cell death pathways [J]. Nat Cell Biol.2001,31(11):255-263.
36.姜山,谢青.内质网应激与细胞凋亡[J].国外医学流行病传染病学分册.2004,31(6)：330-333.
37. Li H, Yuan J Y. Decophering the pathways of life and death [J]. Curr Op in Cell Bio.1999, 11:261-266.
38. Lang R. Rapid colorimetric assay for cell growth and surrvial:Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability [J]. J Immunol Methods.1986,89(2):271-277.
39. Twentyman P R, Luscombe M. A study of some variables in a tetrazolium dye (MTT) bassed assay for cell growth and chemosensitxity [J]. BRJ Cancer.1987,56(3):279-285.
40. Traore A, Baudrimont I. Ambaliou S, et al. DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line [J]. Arch Toxico.2001,75(2)：110-117.
41. Lin XY, Choi MS, Porter AG. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma [J]. Biol Chem.2000,275(51):920-926.
42. Rao RV, Peel A, Logvinova A, et al. Coupling endoplasmic reticulum stress to the cell death program:role of the ER chaperone GRP7 [J]. FEBS Lett.2002(2),514:122-128.
43.黄爱君,黄海燕,庄志雄等.大田软海绵酸对HL-7702肝细胞毒性效应的研究[J].安徽农业科学.2010,38(10)：521-525.
44. Vera Ritz, John Marwitz, Sabine S. Iris Quentin. Contribution of mdrlb-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells [J]. Naunyn Schmiedeberg's Arch Pharmacol.1999,360:116-121.
45. Kerr J F, Wyllie A H. Apoptosis:a basic biological phenomenon with wide ranging locations in tissue kenetics [J]. Journal of Cancer.1972,26:239-245.
46. Creagh E M, Conroy H, Martin S J. Caspase activation pathways in apoptosis and immunity [J]. Immunol Rev.2003,193:10-21.
47. Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase-8 mediates mitochondrial damage in the Fas pathway of apoptosis [J]. Cell.1998,94(4):491-501.
48. Adrain C, Martin S J. The mitochondrial apoptosome:a killer unleashed by the cytochrome seas [J]. Trends Biochem Sci.2001,26(6):390-397.
49.陈洋.DSP等赤潮藻毒素对哺乳类细胞的毒性效应及研究机制[博士学位论文].中国海洋大学：周名江,2008年.
50. Orrenius S. Mitochondrial regulation of apoptotic cell death [J]. Toxi collett.2004, 149(1-3)：19-23.
51. Kimura K, Sasano H, Shimosegawa T, et al. Ultrastructure of cells undergoing apoptosis [J]. Vitam Horm.2000,58:257-266.
52. Hacker G. The morphology of apoptosis [J]. Cell Tissue Res.2000,301(1):5-17.
53. Trump BF, Berezesky IK, Chang SH, et al. The pathways of cell death:oncosis, apoptosis, and necrosis [J]. Toxicol Pathol.1997,25(1):82-86.
54.李沂轩,纪岩文：吖啶橙染色方法检查线索细胞[J].中国皮肤性病学杂志.2005,19(11)：697.
55.廖二元,杨川,戴如春.激光共聚焦显微镜在骨计量学中的应用[J].湖南医科大学学报,2000,25(4)：413-415.
56. Chiba K, Kawakami K, Tohyama K. Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells [J]. Toxicol In Vitro.1998,12:251-258.
57. Steinman RM, Mellman IS, Muller WA, et al. Endocytosis and the recycling of plasma membrane [J]. Cell Biol.1983,96(1):1-27.
58.李振荣,张捷,王天成等.对乳酸脱氢酶同工酶谱的异常区带和增高的乳酸脱氢酶1组织来源的分析[J].北京大学学报(医学版).2004,12(5)：552-553.
59.刘锐克,周军兰,梁建辉.中性红比色法检测大鼠小脑神经瘤活细胞[J].中国药理学与毒理学杂志.2003,17(1)：74-78.
60.孙皎,高桑美代子,佐藤和子等.中性红染色法定量检测牙科材料细胞毒性的研究[J].中国生物医学工程学报.1999,18(1)：118-120.
61. Schwartzman R, Cidlowski J. Apoptosis:the biochemistry and molecular biology of programmed cell death [J]. Endocrine Rev.1993,14:133.
62. Vermes, I. and Haanan, C. Apoptosis and programmed cell death in health and disease [J]. Adv. Clin. Chem.1994,31,177.
63. Majno G, Joris I. Apoptosis, oncosis, and necrosis:An overview of cell death [J]. Am J Pathol.1995,146(1):3-15.
64. Zimmermann K C, Bonzon C, Green D R. The machinery of programmed cell death [J]. Pharmacol Ther.2001,92(1):57-70.
65. Neiman P E, Thomas S J, Loring G. Induction of apoptosis during normal and neoplastic B-cell development in the bursa of Fabricius [J]. Proc Natl Acad Sci U S A.1991,55:5857-5861.
66. Berke G. Debate:the mechanism of lymphocyte-mediated killing Lymphocyte triggered internal target disintegration [J]. Innunol Today.1991,12:396.
67. Suppression of tumo necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control [J]. Proc Natl cad Sci.1997,56:57-62.
68. Khoshnan A, Tindell C. The NF-kappa Bcascade is important in Bclxl expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4 lymphocytes [J]. Immunol.2000,165:1743-1754.
69. Stehlik C, Martin R, Kumabashiri, et al. Nuclear factor(NF)-kappa-B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis [J]. Exp Med.1998,58:211-216.
70. Asso-Bonnet M, Feuillard J. Relationship between kappa B alpha constitutive expression, TNF alpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells [J]. Oncogene.1998,59(17)：1607-1615.
71. Lee K Y, Chang W, Qiu D, et al. PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells [J]. Biol Chem.1999,60:451-455.
72. Sugiyama H, Savill J S. Selective sensitization to tumor necrosis factor alpha induced apoptosis by blockade of NF kappa B in primary glomerular mesangial cells [J] Biol Chem.199961：532-537.
73. Shao R, Karunagaran D, Zhou B. P, et al. Inhibition of nuclear factor-kappa B activity is involved in ElA-mediated sensitization of radiation-inducedapoptosis [J]. Biol Chem.1997,62：739-742.
74. Kajino S, Suganuma M, Teranishi F et al. Evidence that de novo protein synthesis is dispensable for anti-apoptotic-effects of NF-kappaB [J]. Oncoaene.2000,63:233-239.
75. Javelaud D, Besancon F. NF-kappa B activation results in rapid inactivation of JNK in TNF alpha treated Ewing sarcoma cells:a mechanism for the anti-apoptotic effect of NF-kappa B [J]. Oncoaene.200164:365-372.
76. Martindale J L, Holbrook N J. Cellular response to oxidative strees:signaling for suicide and survival [J]. Cell Physio.2002,192(1):1-15.
77. Dypbukt J M, Ankarcrona M, Burkitt M et al. Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines [J]. Biol Chem.1994,269(48):553-560.
78. Lin, X Y, Choi M S, Porter A G. Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-gamma [J]. Biol Chem.2000,275:920-926.
79. Vinella D, D'Ari R. Overview of controls in the Escherichia coli cell cycle [J]. Bioessays.1995,17(6)：527-536.
80. Norbury C, Nurse P. Animal cell cycles and their control [J]. Ann Rev Biochem.1992,61: 441-470.
81. Bergrnann M. W, Loser P, Dietz R, et al. Effect of NF kappa B Inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes [J]. Mol Cell Cardiol.2001,65:1223-1232.
82. Klumpp S, Rieglstein J. Serine/threonine protein phosphatases in apoptosis [J]. Curr Opin Pharmaco.2002,2(4):458-462.
83. Liu J P, Schmid P C. Protein serine/threonine phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 tomodulate its transcriptional and apoptotic activities [J]. Oncogene. 2006,25(21):3006-3022.