【作者】 孙静秋；

【导师】 许燕；

【作者基本信息】 上海师范大学 ， 动物学， 2008， 硕士

【摘要】 南美白对虾（Litopenaeus vannamei）是世界产量最高的三大虾种之一，引入国内后正逐步走向淡水化养殖，随着该养殖业的迅速发展,越来越多的对虾疾病给养虾业造成了严重的经济损失，其中病毒是对虾暴发性流行病发生的主要病原因子。目前国内外学者对南美白对虾主要进行免疫学方面的研究，探讨提高机体抗病力的各种免疫学途径，并力图找到便于监测其免疫机能状态的生化指标。因此，一些与免疫相关的同工酶如ACP、AKP、SOD、POD以及PPO等越来越受到重视。综合研究与南美白对虾免疫作用相关的多种同工酶，对于了解对虾病毒性疾病的防治与诊断有重要的理论参考价值。1南美白对虾体内五种同工酶酶谱的研究本文利用聚丙烯酰胺凝胶垂直平板电泳技术以及同工酶染色技术，重点对南美白对虾成体的复眼、肝脏、心脏、鳃和肌肉等五种组织中与免疫相关的ACP、AKP、SOD、POD和PPO等五种同工酶进行研究。比较健康对虾与患病毒性疾病的对虾各同工酶酶谱表型变化，结果显示五种同工酶在南美白对虾体内均有分布，且同一种同工酶在不同组织中表达的条带数量以及酶活性均存在组织特异性：除SOD在心脏中的表达最强外，其余四种同工酶均在肝脏中的表达最强；而复眼中的ACP、AKP、SOD以及肌肉中POD和PPO表达最弱。感染病毒后，病虾体内的生理代谢发生明显紊乱，这五种同工酶的酶谱发生了明显变化。ACP、AKP、POD和PPO这四种同工酶在肝脏和鳃中出现一些特异性酶带的缺失或酶活性减弱的现象。如病虾的ACP酶谱在肝脏缺失了2条特异性酶带ACP-5、9，在健康对虾肝脏中染色较深的ACP-2、3在染病后其酶活性显著降低，鳃中缺失1条特异性酶带ACP-8，且2条原先染色较深带较宽的条带ACP-1、6的酶活明显减弱；病虾的AKP同工酶酶谱在原先健康体的鳃组织中所特有的3条酶带AKP-4、9、12全部缺失，且染色较深的AKP-1酶活性减弱，肝脏中原先有表达的6条特异性酶带中有1条AKP-8缺失，酶活性明显减弱的条带有AKP-1、2；病虾的POD同工酶酶谱在原先健康对虾的肝脏中有表达的4条特异性酶带POD-1、2、3、8全部缺失，鳃中原先1条染色较深酶带较宽的条带POD-4缺失；病虾的PPO同工酶酶谱在原先健康对虾肝脏组织中有表达的2条特异性酶带中缺失一条PPO-1，它也是PPO同工酶中染色最深带最宽的条带，鳃中原先染色较深的PPO-2缺失。而病虾的SOD同工酶酶谱则在心脏和鳃中呈现下降趋势，表现为心脏中缺失1条特异性酶带SOD-5；鳃中缺失1条染色较深的酶带SOD-3。此外ACP、AKP、POD和PPO这四种同工酶在心脏中则出现酶带增加或酶活性增强的现象。如ACP、POD和PPO在心脏中各新增了1条酶带；AKP在心脏中新增3条。而SOD则在肝脏组织中呈现酶活性增强的趋势，虽然未出现新增带，但是其原先表达的3条酶带中的2条染色明显加深条带也加宽。总之这五种同工酶的表型在健康虾和病虾之间存在明显的差异，尤其是在肝脏和心脏中的变化最为明显，说明这二个组织中五种同工酶的特异性变化可以作为研究南美白对虾病毒病的一种辅助生理生化指标。2南美白对虾体内五种同工酶的酶细胞化学定位的研究运用电镜酶细胞化学技术对南美白对虾体内肝脏、肌肉、心脏、复眼和鳃等五种组织的ACP、AKP、SOD、POD和PPO的酶细胞化学定位进行了研究，并与感染病毒的南美白对虾体内五种组织中ACP、AKP、SOD、POD和PPO的细胞化学定位进行比较。结果显示健康虾体内ACP多定位于细胞核、内质网和溶酶体等不同细胞器中，肝脏中的ACP分布较广泛，还定位于肝脏细胞内的微绒毛、脂滴周围、线粒体以及细胞内膜；AKP常见于细胞核、细胞核膜、内质网、细胞膜、次级溶酶体，还定位在肝细胞中的脂滴、微绒毛、细胞内膜、线粒体以及鳃细胞中的线粒体膜上；SOD酶只定位于各组织细胞的线粒体膜、细胞质中，以及肝细胞的脂滴周围；POD酶主要定位于心脏、鳃和肝脏组织细胞的过氧化物酶体内，肝细胞中脂滴周围也有POD的定位，而在复眼和肌肉中的过氧化物酶体很少有POD的分布；PPO主要以酚氧化酶原形式分布于肝脏细胞的细胞质中，心脏、肌肉细胞中的PPO阳性颗粒较少。当病毒感染后，各组织细胞内出现了明显的病理性结构变化：内质网（肌质网）、线粒体肿胀扩张，呈空泡状；细胞质混浊，其中出现大量的髓样小体；肌纤维排列疏松、散乱，有明显的断裂；脂滴呈小颗粒状分散在细胞质中，微绒毛稀少；细胞核形状不规则，偶尔能见到核溶解、核固缩现象等等。同时各类同工酶的酶细胞化学定位也发生了变化，表现为：ACP定位只集中于髓样小体、线粒体外膜、次级溶酶体、小颗粒状脂滴周围以及细胞质；AKP集中在一些膜性结构上，如内质网、髓样小体、线粒体膜，且阳性反应减弱，除此之外胞质中也能发现AKP阳性颗粒；原先在胞质中均有定位的SOD同工酶，现只存在于肝细胞中，另外还发现线粒体越是受损严重的部位，SOD阳性反应也越强，尤其是在心脏以及鳃中，不仅在线粒体外膜有SOD阳性反应，而且其致密化的基质内也发现SOD的定位；POD仍出现在胞质中的过氧化物酶体中，数量减少，且部分过氧化物酶体解体；肝脏组织中几乎未见PPO阳性，而心脏中除了定位到酚氧化酶原之外，在基质致密化的线粒体内部也定位到PPO酶活性，其余三种组织中，PPO均定位在酚氧化酶原以及细胞质中大量出现的髓样小体上。结果表明ACP、AKP、PPO均有定位的溶酶体在南美白对虾免疫防御过程中起着破坏并且消化衰退的膜性结构的作用，SOD、POD在清除机体内过量O₂^-方面起着重要的作用。从感染病毒后南美白对虾体内各组织中的ACP、AKP、SOD、POD和PPO酶细胞化学定位的规律性变化，可以认为其ACP、AKP、SOD、POD和PPO在对虾的生长和免疫反应中发挥着积极的作用。更多还原

【Abstract】 Litopenaeus vannamei is one of the most important farmed products in the world.The introduction of L. vannamei in shrimp farming is step by step from seawater tofreshwater. With rapid growth of shrimp farming, more and more diseased problems ofshrimps cause a major loss in shrimp aquaculture. The virus is a main pathogenic factorin explosive epidemic shrimp disease. Presently, experts research on immunology of L.vannamei in order to finding several ways to improve the immunity against disease.And they try to find a biochemical marker of immunological mechanism. Therefore，theimmune-related enzymes such as phosphatase (ACP), alkaline phosphatase (AKP),superoxide dismutase (SOD), peroxidase (POD), polyphenoloxidase (PPO) and so onhave been paid more attention. Research on these enzymes in L. vannamei is importantreference value in theory to understand the technology of pathogens diagnosis, thetreatment of disease problems and especially the prevention studies on shrimp.1 Study on Electrophoretograms of Five Isozymes of LitopenaeusvannameiVertical polyacrylamide gel electrophoresis (PAGE) and biochemical staining wereused to analyze five isozymes including ACP, AKP, SOD, POD and PPO in the eye,liver, heart, gill and muscle of L. vannamei. The electrophoretic patterns of theisozymes showed differences between the healthy shrimps and the diseased shrimpsinfected with virus. The results showed that the five isozymes were all expressed infive kinds of tissues of L. vannamei, and activities of the five isozymes were tissuespecific. Except SOD was the highest in heart of L. vannamei. The activities of theother isozymes were all the highest in liver. After shrimps were infected with virus,physiological metabolism in diseased shrimps was apparently beginning to fail. Thephenotypes of five isozymes had some obviously changes, and some characteristicbands of ACP, AKP, POD and PPO decreased or got weaker in liver and gill. ACPdecreased 2 characteristic bands in liver in diseased shrimps. Furthermore, ACPdecreased 1 characteristic band in gill, and 2 bands got weaker. AKP decreased 1 and 3characteristic bands in liver and gill respectively. POD decreased 4 characteristic bands in liver, and decreased 1 band which was deeper and thicker band in gill of healthyshrimps. PPO decreased 1 characteristic band which was the deepest and thickest bandin healthy shrimps in liver, and in gill also decreased 1 band. But on the downtrend ofSOD activity was in heart and gill. SOD decreased 1 band in heart and gill respectivelyin diseased shrimps. Furthermore, higher activities of ACP, AKP, POD and PPO weredetected in heart of diseased shrimps. ACP, POD and PPO presented 1 new bandrespectively, and AKP presented 3 new ones in heart. Higher activity of SOD wasdetected in liver. Although there was no new band of SOD in heart, 2 bands whichwere detected in healthy shrimps got deeper and thicker. Due to theelectrophoretogram of fiver isozymes changed most obviously in liver and heart ofdiseased shrimps with virus, the shrimp isozymes in liver and heart can be used asbiochemical marker for diagnosis of L.vannamei with virus.2 Study on Cytochemical Location of Five Isozymes of LitopenaeusvannameiThe activities and location of ACP, AKP, SOD, POD and PPO in the liver, muscle,heart, gill and eye of L. vannamei were studied with enzyme cytochemical techniqueof electronic microscope, compared it with the infected shrimp. The results showedthat the ACP activities were mainly deposited in nucellar cell, endoplasmic reticulumand lysosome, and in liver ACP were detected in microvilli, around lipid droplet,mitochondrium and cellular endomembrane. AKP activities were mainly deposited innucellar cell, nuclear membrane, endoplasmic reticulum, cellular membrane andsecondary lysosome. And in liver AKP were detected in microvilli, around lipiddroplet, mitochondrium and cellular endomembrane, and mitochondrial membrane ingill of the healthy shrimps. The SOD activity was site in mitochondrium, cytoplasm incells and around lipid droplet. And the POD and PPO activities were site in specificorganelle respectively: peroxisome and prophenoloxidase. In infected shrimp withvirus, the pathological changes were obviously in tissues and cells. These changeswere showed as follows: The dilatation of endoplasmic reticulum (sarcoplasmicreticulum) and mitochondrium became small vacuoles. Cytoplasm was concentrationand turbidity. More myelin figures appeared in cytoplasm of cells. Muscle fiberbecame loose, very relaxed and obviously ruptured. Lipid droplet was isolated smallgranule in the cytoplasm. Microvilli were fewer. And Nucellar cell became abnormal.At the same time, the activities site of five enzymes had changes in cells. ACP positivereaction granules were only observed in myelin figure, mitochondrial membrane, secondary lysosome, around small lipid droplet and cytoplasm. AKP positive reactiongranules were site in endoplasmic reticulum, myelin figure, mitochondrial membraneand cytoplasm. SOD positive reaction was found in mitochondrial membrane whichwas damaged seriously and densification of mitochondrial matrix. But there were fewSOD positive granules in the cytoplasm. POD positive reaction was still found inperoxisome which decreased in the number. And part of peroxisome was disaggregated.The activity site of PPO was not only in prophenoloxidase, but also in myelin figureand densification of mitochondrial matrix. But PPO positive activity wasn’t observedin liver. The results were indicated that lysosomes in which ACP, AKP and PPOactivities were located destroyed and digested membrane structure in immunologicaldefense of L. vannamei. SOD and POD played an important role in eliminatingexcessive superoxide anion in shrimps. From the regularity changes of cytochemicallocation of ACP, AKP, SOD, POD and PPO of L. vannamei, we think that the fiveisozymes have positive effects on the growth and immunoreaction of L. vannamei.更多还原