亚硒酸钠胁迫下斑点叉尾鮰脑组织差异蛋白质组研究
摘要
硒是动物和人必须的微量元素之一,具有抗氧化、抗应激、加强机体免疫力、抗肿瘤等重要作用。本论文以斑点叉尾鮰为研究对象,在开展亚硒酸钠对斑点叉尾鮰的急性毒性试验、半致死浓度协迫下脑组织病理观察的基础实验上开展脑组织差异蛋白质组的初步研究。
第一部分开展亚硒酸钠对斑点叉尾鮰幼鱼的急性毒性试验,结果表明：试验组斑点叉尾鮰易受惊扰,身体失去平衡,或是侧游、或是仰游、或是翻滚。经统计学分析得到亚硒酸钠对鮰鱼96h半致死浓度为35.59mg/L
第二部分依据急性毒性试验分析结果,取经过半致死浓度35.59mg/L浸泡96h后还存活的鱼小脑进行石蜡组织切片和电镜观察。组织切片结果表明：对照组小脑皮质颗粒层细胞排列均匀,颗粒层结构完整,呈正常的圆形；实验组小脑皮质颗粒层区域疏松、水肿,呈现空网状,颗粒细胞崩解消失,部分细胞失去正常的圆形,出现菱角,呈三角形或者不规则形状。电镜结果表明：对照组小脑皮质细胞内有丰富的细胞器,线粒体丰富,嵴板排列规整,髓鞘结构完整；实验组小脑皮质细胞质水肿,细胞质内细胞器明显减少,线粒体水肿,双层膜结构坍塌,嵴溶解消失,有的线粒体呈空泡状,仅见线粒体的轮廓。病理观察表明亚硒酸钠对斑点叉尾鮰脑组织有一定的毒性损伤。
第三部分同样取经过半致死浓度35.59mg/L浸泡96h后还存活的鱼脑组织进行蛋白质组学研究。首先通过优化双向凝胶电泳各方面条件后建立一套适合于斑点叉尾鮰脑组织双向电泳体系,分析双向电泳图谱找出差异蛋白点。结果表明：对照组分离出蛋白质点1024±25个,实验组为998±20个,对照组与实验组共有9个差异表达的蛋白质点。将差异蛋白质点进行生物质谱分析鉴定,并通过对NCBInr和EST数据库检索得到9种蛋白质,其中3种蛋白质为peroxiredoxin-2、aldose reductase和carbonic anhydrase,有望作为研究斑点叉尾鮰脑组织毒性损伤后的指示蛋白质。
Selenium is an essential trace element for animals and human being. It serves as antioxidant, anti-stress, immunity enhancement, anti-tumor, etc. In this thesis, channel catfish as experimental research object, Conduct sodium selenite acute toxicity tests half lethal concentration intimidation brain pathological observation and brain differences proteomics research.
In the first part, channel catfish was considered as the experimental research watched. Handling with sodium selenite, the channel catfish's behaviors were surveyed. The results showed that:the experimental samples were vulnerable to disturb, and lost their balance (showed swim in side directions, or backstroke, or rolling). The statistical analysis showed that the sodium selenite on the 96h half lethal concentration of 35.59mg/L.
In the second part, based on the statistical analysis results of acute toxicity test, the live channel catfish cerebellum after half lethal concentration (35.59mg/L,96h) exposure test were taken and made into paraffin sections. The sections were observed with transmission electron microscopy (TEM). The results of paraffin sections showed that:the control group cerebellar cortex granular cells arranged regularly, granular layer structures were in integrity, showed in normal round shapes. The experimental group cerebellar cortex loose granular layer, edema, showing an empty net, granular cell collapse disappeared, some cells loss of normal round, triangular or irregular shape. TEM results show that:the control group within the cerebellar cortex is rich in organelles, mitochondria rich in cristae arranged in regular panels, structural integrity of myelin sheath. The experimental group cerebellar cortical cytoplasm edema, decreased cytoplasmic organelles, mitochondrial swelling, collapse of membrane structure, cristae dissolution disappeared, and some mitochondrial vacuolization, only to see the outline of mitochondria. Pathological observation shows that sodium selenite has certain toxic injury to the brain of channel catfish.
In the third part, the survivals'brains which had been dealt with half lethal concentration 35.59mg/L exposed after 96h have been taken for the proteomics research. By optimizing 2-DE, a set of conditions for channel catfish brain dimensional electrophoresis system was obtained. Using ImageMaster software, the 2-DE maps of brain tissue from both the control group and the experimental group were analyzed, and the differences in protein spots were identified. The results showed that:1024±25 protein spots from the control group and 998±20 protein spots form the experimental group were isolated. The difference protein point was separate identification of biological mass spectrometry. And we get 9 proteins through to the NCBInr and EST databases retrieving. The three proteins peroxiredoxin-2, aldose reductase and carbonic anhydrase among it may be biomarkers of selenium strengthening the body antioxidant immune system proteins.
第一部分开展亚硒酸钠对斑点叉尾鮰幼鱼的急性毒性试验,结果表明：试验组斑点叉尾鮰易受惊扰,身体失去平衡,或是侧游、或是仰游、或是翻滚。经统计学分析得到亚硒酸钠对鮰鱼96h半致死浓度为35.59mg/L
第二部分依据急性毒性试验分析结果,取经过半致死浓度35.59mg/L浸泡96h后还存活的鱼小脑进行石蜡组织切片和电镜观察。组织切片结果表明：对照组小脑皮质颗粒层细胞排列均匀,颗粒层结构完整,呈正常的圆形；实验组小脑皮质颗粒层区域疏松、水肿,呈现空网状,颗粒细胞崩解消失,部分细胞失去正常的圆形,出现菱角,呈三角形或者不规则形状。电镜结果表明：对照组小脑皮质细胞内有丰富的细胞器,线粒体丰富,嵴板排列规整,髓鞘结构完整；实验组小脑皮质细胞质水肿,细胞质内细胞器明显减少,线粒体水肿,双层膜结构坍塌,嵴溶解消失,有的线粒体呈空泡状,仅见线粒体的轮廓。病理观察表明亚硒酸钠对斑点叉尾鮰脑组织有一定的毒性损伤。
第三部分同样取经过半致死浓度35.59mg/L浸泡96h后还存活的鱼脑组织进行蛋白质组学研究。首先通过优化双向凝胶电泳各方面条件后建立一套适合于斑点叉尾鮰脑组织双向电泳体系,分析双向电泳图谱找出差异蛋白点。结果表明：对照组分离出蛋白质点1024±25个,实验组为998±20个,对照组与实验组共有9个差异表达的蛋白质点。将差异蛋白质点进行生物质谱分析鉴定,并通过对NCBInr和EST数据库检索得到9种蛋白质,其中3种蛋白质为peroxiredoxin-2、aldose reductase和carbonic anhydrase,有望作为研究斑点叉尾鮰脑组织毒性损伤后的指示蛋白质。
Selenium is an essential trace element for animals and human being. It serves as antioxidant, anti-stress, immunity enhancement, anti-tumor, etc. In this thesis, channel catfish as experimental research object, Conduct sodium selenite acute toxicity tests half lethal concentration intimidation brain pathological observation and brain differences proteomics research.
In the first part, channel catfish was considered as the experimental research watched. Handling with sodium selenite, the channel catfish's behaviors were surveyed. The results showed that:the experimental samples were vulnerable to disturb, and lost their balance (showed swim in side directions, or backstroke, or rolling). The statistical analysis showed that the sodium selenite on the 96h half lethal concentration of 35.59mg/L.
In the second part, based on the statistical analysis results of acute toxicity test, the live channel catfish cerebellum after half lethal concentration (35.59mg/L,96h) exposure test were taken and made into paraffin sections. The sections were observed with transmission electron microscopy (TEM). The results of paraffin sections showed that:the control group cerebellar cortex granular cells arranged regularly, granular layer structures were in integrity, showed in normal round shapes. The experimental group cerebellar cortex loose granular layer, edema, showing an empty net, granular cell collapse disappeared, some cells loss of normal round, triangular or irregular shape. TEM results show that:the control group within the cerebellar cortex is rich in organelles, mitochondria rich in cristae arranged in regular panels, structural integrity of myelin sheath. The experimental group cerebellar cortical cytoplasm edema, decreased cytoplasmic organelles, mitochondrial swelling, collapse of membrane structure, cristae dissolution disappeared, and some mitochondrial vacuolization, only to see the outline of mitochondria. Pathological observation shows that sodium selenite has certain toxic injury to the brain of channel catfish.
In the third part, the survivals'brains which had been dealt with half lethal concentration 35.59mg/L exposed after 96h have been taken for the proteomics research. By optimizing 2-DE, a set of conditions for channel catfish brain dimensional electrophoresis system was obtained. Using ImageMaster software, the 2-DE maps of brain tissue from both the control group and the experimental group were analyzed, and the differences in protein spots were identified. The results showed that:1024±25 protein spots from the control group and 998±20 protein spots form the experimental group were isolated. The difference protein point was separate identification of biological mass spectrometry. And we get 9 proteins through to the NCBInr and EST databases retrieving. The three proteins peroxiredoxin-2, aldose reductase and carbonic anhydrase among it may be biomarkers of selenium strengthening the body antioxidant immune system proteins.
引文
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