【作者】 张胜根；

【导师】 邢克智； 孙敬锋；

【作者基本信息】 天津农学院 ， 水产养殖学， 2011， 硕士

【摘要】 1、研究点带石斑鱼不同组织器官中一氧化氮(NO)含量和不同类型一氧化氮合酶(NOS)活性,采用Griess试剂法测定NO含量,采用化学比浊法测定NOS的活性。研究结果表明：诱导型一氧化氮合酶(iNOS)在头肾组织中活性最高,依次为肌肉、肝脏、脾脏、肠、胃、胰脏、眼睛、脑、心脏、鳃、中肾。结构型一氧化氮合酶(cNOS)在脑组织中活性最高,依次为眼睛、肠、肝脏、胃、肌肉、头肾、鳃、胰脏、中肾、心脏、脾脏。NO含量在脾脏中最高,依次为肝脏、头肾、中肾、心脏、胰脏、肠、脑、眼睛、胃、鳃、肌肉、血清。2、通过使用免疫组织化学SP(链霉菌抗生物素蛋白-过氧化物酶连结法)法以及NADPH-d(还原型尼克酰胺腺嘌呤二核苷酸脱氢酶)酶组织化学染色法测定神经型一氧化氮合成酶(nNOS)在点带石斑鱼各组织中的分布及定位。酶免疫组织化学定位结果表明,nNOS阳性结果分布在间脑中的神经元,大脑和中脑的神经元和神经纤维中,小脑的颗粒细胞、阳性纤维和普肯野细胞；视网膜的视锥视杆细胞层,外核层,外网层、内核层、内网层、节细胞层和视神经纤维层；中肾组织的近曲小管和远曲小管；头肾组织的前肾间组织和网状细胞；心脏组织的神经纤维和心肌纤维；脾脏的血窦中的红细胞和脾索的网状细胞；肠组织的纹状缘、神经元和基膜；肝脏中的肝细胞和中央静脉壁；肌肉组织中的肌纤维处；鳃丝的上皮细胞,沿动脉血管延伸的神经纤维和红细胞。NADPH-d酶组织化学染色结果表明,NOS分布在间脑神经元、大脑和中脑的神经元和神经纤维、小脑神经元和颗粒细胞、神经纤维和普肯野细胞；视网膜中视锥视杆细胞层,外核层,外网层,内核层、内网层、节细胞层和视神经纤维层；中肾的近曲小管、远曲小管,神经纤维和血管壁中；头肾的前肾间组织、动脉血管的神经纤维、神经元和网状细胞；心脏的心肌纤维和神经纤维；脾脏的红细胞和网状细胞中；肠组织中的纹状缘、神经元和肌间神经纤维；肝脏的肝细胞、中央静脉、神经纤维,枯否氏细胞,小叶间动脉,小叶间静脉和小叶间胆管；肌肉组织的肌纤维处；鳃丝的上皮细胞,沿动脉血管延伸的神经纤维、红细胞、泌氯细胞和柱细胞。利用Western Blot技术对各组织中的nNOS蛋白分子量进行测定,结果表明,脑,眼睛,头肾、中肾、心脏、鳃和胃中有三条带,对应分r量分别为130kD、120 kD和80kD,而在肠、脾脏、肝脏、胰脏和肌肉中有两条带,对应分子量120kD和80kD。3、研究刺激物对点带石斑鱼血清和头肾NO含量和iNOS活性的影响,刺激物包括：脂多糖(LPS)、酵母多糖(zymosan)和嗜水气单胞菌。体外培养头肾巨噬细胞,利用LPS刺激头肾巨噬细胞,检测NO含量和iNOS活性,结果表明,LPS能诱导巨噬细胞iNOS的表达,催化产生大量的NO,L-单甲基-精氨酸(L-NMMA)和L-硝基-精氨酸甲脂(L-NAME)能抑制NO的产生,iNOS最大活性出现在刺激后的12h,NO的最大产量出现在刺激后的24h。利用zymosan和嗜水气单胞菌对点带石斑鱼进行腹腔注射,检测头肾和血清中的NO含量和iNOS活性,结果表明,iNOS最大活性同样出现在刺激后的12h,NO的最大产量也出现在刺激后的24h。这证明体内和体外的iNOS活性增长和NO含量的增加具有同样的趋向性,体内和体外的iNOS活性增长和NO含量的增加具有同样的时效性。4、利用免疫组化sp法以及NADPH-d酶组织化学染色法测定诱导型一氧化合酶(iNOS)在点带石斑鱼各组织中的分布及定位。酶免疫组织化学定位结果表明,iNOS主要存在于脑的阳性神经元、神经纤维和小脑浦肯野细胞中；视网膜的视锥视杆细胞层,外网层,内核层、内网层、竹细胞层和视神经纤维层中;中肾组织中近曲小管、远曲小管和巨噬细胞中；头肾的前肾间组织、巨噬细胞、中性粒细胞和淋巴细胞中;心脏中心肌纤维；脾脏的巨噬细胞、中性粒细胞和淋巴细胞中；肠中纹状缘和巨噬细胞；肌肉组织中肌纤维处；肝脏的肝细胞和枯否氏细胞中；鳃的神经纤维、上皮细胞和血细胞中。NADPH-d酶组织化学染色结果表明,NOS分布在脑的神经元、神经纤维和小脑浦肯野细胞；视网膜的视锥视杆细胞层、外核层、外网层、内核层、内网层、节细胞层和视神经纤维层；中肾的近曲小管、远曲小管和巨噬细胞中：头肾的前肾间组织、淋巴细胞、巨噬细胞和中性粒细胞；心脏中心肌纤维处；脾脏中巨噬细胞、中性粒细胞和淋巴细胞中；肠组织中纹状缘和巨噬细胞；肌肉中肌纤维处；肝脏的肝细胞和枯否氏细胞中；鳃组织中神经纤维,上皮细胞和泌氯细胞中。利用Western Blot技术对各组织中的iNOS蛋白分子量进行测定,试验结果显示,iNOS在脑、眼睛、肌肉、鳃、头肾、胃、肝脏、脾脏、中肾、肠中有120 kD和80 kD两条带,在心脏只有一条带80kD,而在胰脏中没有带出现。更多还原

【Abstract】 1. To study nitric oxide production and nitric oxide synthase activity in the organizations of Epinephelus malabaricus. Griess Reagent was used to determinate the nitric oxide (NO) production, and the method of chemical colorimetry was used to measure the nitric oxide synthase (NOS) activity. Inducible NOS (iNOS) activity was highest in the head-kidney, followed by the muscle, liver, spleen, intestine, stomach, pancreas, eye, brain, heart, sill and kidney. The constitutive NOS (cNOS) activity was highest in the brain, followed by the eye, intestine, liver, stomach, muscle, head-kidney, sill, pancreas, kidney, heart and spleen. The spleen was found to have the highest level of NO production, followed by the liver, head-kidney, kidney, heart, pancreas, intestine, brain, eye, stomach, sill, muscle and serum.2. Immunohistochemical localization SP (streptavidin-perosidase) method and NADPH-diaphorase (nicotinamide adenine dinucleotide hydrogen phosphate-diapho-rase) staining were used to study distribution and localization of neuronal NOS (nNOS) in the organizations of Epinephelus malabaricus. The results of enzyme immunohistochemical localization showed that the positive results of nNOS were located in neurons of the diencephalons. in neurons and nerve fibers of brain and mesencephalon. in granular cells, nerve fibers purkinje cells of the cerebellum; in the retina, layer of cones and rods, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cell and layer of optic nerve fibers; in the kidney, proximal convoluted tubule and distal convoluted tubule; in the head kidney, anterior interrenal tissues and reticular cells; in the heart, nerve fibres and myocardial fibers; in the spleen, red cells of splenic sinusoids and the reticular cells of splenic cord; in the intestine, striated border, neurons and basement membrane; in the liver, liver cells and central vein; muscle fibres of the muscle; in the gill, epithelial cells of the filament, nerve fibers run along vein blood and red cells. The results of NADPH-d histochemical staining showed that NOS were distributed in neurons of the diencephalons, neurons and nerve fibers of brain and mesencephalon, granular cells, nerve fibers purkinje cells of the cerebellum; in the retina. layer of cones and rods, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cells and layer of optic nerve fibers; in the kidney, proximal convoluted tubules, distal convoluted tubules, nerve fibres and vessel wall; in the head kidney, anterior interrenal tissues, nerve fibers of arteries, neurons and reticular cells; in the heart, nerve fibers and myocardial fibers; the red cells and the reticular cells of the spleen; in the intestine, striated border, neurons and myenteric nerve fibers; in the liver, liver cells, nerve fibers, kupffer cells, interlobular artery, interlobular vein and interlobular bile duct; muscle fibres of the muscle; in the gill, epithelial cells of the filament, nerve fibers run along vein blood, chloride cells and red cells. Western Blot was used to analysis protein molecular weight of nNOS in organizations, the results show that there were three belts in the brain, eye, head kidney, kidney, heart, gill and stomach, respectively, corresponding molecular weight were 30 kD,120 kD and 80 kD. However, there were two belts in the intestine, spleen, liver, pancreas and muscle, respectively, corresponding molecular weight were 120 kD and 80 kD.3. To study the effect of irritants about nitric oxide production and nitric oxide synthase activity in the serum and head kidney of Epinephelabaricus. Irritants include:lipopolysaccharide (LPS), zymosan and Aeromonas hydrophila. The macrophages of head kidney were cultured in vitro, to study NO production and iNOS activity, the macrophages of head kidney were stimulated by LPS, the results show that iNOS of macrophages could be induced by LPS, a large amounts of NO could be catalyzed by iNOS. however, NG-minomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine methylester (L-NAME) could inhibit the production of NO. The most of iNOS activity appeared at 12 hour after stimulated, the most of NO production appeared at 24 hour after stimulated. To study NO production and iNOS activity in the serum and head kidney, zymosan and Aeromonas hydrophila were injected in abdominal cavity of Epinephelus malabaricus, the results show that the most of iNOS activity also appeared at 12 hour after stimulated, the most of NO production also appeared at 24 hour after stimulated. The study proved that growth of iNOS activity and increase of NO production had the same tendency in vivo and in vitro, growth of iNOS activity and increase of NO production had the same timeliness in vivo and in vitro. 4. Immunohistochemical localization SP method and NADPH-diaphorase staining were used to study distribution and localization of inducible NOS (iNOS) in the organizations of Epinephelus malabaricus. The results of enzyme immunohistochemical localization showed that iNOS were distributed in neurons and nerve fibers of the brain, purkinje cells of the cerebellum; in the retina, layer of cones and rods, outer plexiform layer, inner nuclear layer, inner plexiform layer, layer of ganglion cells and layer of optic nerve fibers; in the kidney, proximal tubules, distal convoluted tubules and macrophages; in the head kidney, anterior interrenal tissues, macrophages and neutrophils; myocardial fiber of the heart; in the spleen, the macrophages, neutrophils and lymphocytes; in the intestine, the striated border and macrophages; muscle fibres of the muscle; in the liver, liver cells and kupffer cells; in the gill, nerve fibers, epithelial cells and red cells. The results of NADPH-d histochemical staining showed that NOS were distributed in neurons and nerve fibers of the brain, purkinje cells of the cerebellum; in the retina, NOS were distributed in the cone and rod layer, outer nuclear layer, outer plexiform layer, inner nuclear layer, the network layer, ganglion cell layer and optic nerve fiber layer; in the kidney, the proximal tubules, distal convoluted tubules and macrophages; in the head kidney, anterior interrenal tissues, macrophages and neutrophils; myocardial fiber of the heart; in the spleen, macrophages. neutrophils and lymphocytes; striated border and macrophages of the intestine; in muscle fibres of muscle; liver cells and kupffer cells of the liver; in the gill, nerve fibers, epithelial cells and chloride cells. Western Blot was used to determine protein molecular weight of iNOS in organizations, the results show that there were two belts in the brain, eye, muscle, gill, head kidney, stomach, liver, spleen, kidney and intestine, respectively,120 kD and 80 kD. There was one belt in the heart,80 kD. however, there was no belt appeared in the pancreas.更多还原