大田软海绵酸（Okadaic Acid，OA）对小鼠胚胎细胞3T3的影响

【作者】 张晓旭；

【导师】 王振龙； 王仁君；

【作者基本信息】 曲阜师范大学 ， 动物学， 2011， 硕士

【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。更多还原

【Abstract】 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 OA5. 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 cellWhen 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 microscopeResults 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 microscopyThe 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 experimentsThe 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 kitThe 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 levelsThe 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 activityThe 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.更多还原