【作者】 刘红柏；

【导师】 华育平； 孙大江；

【作者基本信息】 东北林业大学 ， 野生动物保护与利用， 2004， 博士

【摘要】 史氏鲟是黑龙江特有的经济鱼类，目前已成为重要的养殖品种。对史氏鲟免疫学的研究是养殖过程中疾病的防治以及免疫技术应用的基础。本文采用组织学、生物化学及免疫学等方法对史氏鲟的免疫学及相关因子进行了研究，同时也探讨了史氏鲟免疫系统在免疫应激(包括疾病感染和免疫药物刺激)中的变化。 血细胞是重要的免疫相关细胞。光镜下可以看到史氏鲟的外周血中有网织红细胞和处于分裂状态的红细胞，含有四种类型白细胞，分别为淋巴细胞、粒细胞、血栓细胞和单核细胞。在电子显微镜下观察：红细胞中具有少量的细胞器；淋巴细胞结构典型：单核细胞较粒细胞稍小且具有较多线粒体；血栓细胞具有梭形和圆形两种，胞质较少，其中梭形的血栓细胞胞质几乎透明；史氏鲟的粒细胞有两种不同形态的电子密度高及电子密度低的颗粒。 鱼类的血清中含有多种蛋白，其中的抗体构成了—个特异性的体液防御体系。对2~+龄史氏鲟，3~+龄中华鲟及3龄达氏鳇的血清蛋白及血清免疫球蛋白进行了研究。血清蛋白分析的结果表明：史氏鲟、达氏鳇及中华鲟分别有11、8及7条血清蛋白谱带；血清蛋白相似系数的结果表明中华鲟和史氏鲟的相似性要高于达氏鳇和史氏鲟；三种鲟鱼的血清蛋白及其组分的含量也存在差别。采用凝胶层析的方法，纯化制备了健康非免疫状态下这三种鲟鱼的血清免疫球蛋白IgM，采用蛋白免疫印迹验证了所提纯物质IgM，同时检测出其重链池具有一定的抗体活性而轻链的检测结果呈阴性。通过PAGE的方法测定出：史氏鲟，中华鲟和达氏鳇IgM的相对分子量分别为867KD，896KD和924KD；这三种鱼IgM的重链分子量均为88KD，都具有29KD的轻链，其中达氏鳇还另有一分子量约为26KD的轻链蛋白。对这三种鲟鱼IgM的同源性分析表明它们的血清Ig具有同源性，与相互之间的兔抗Ig都有免疫沉淀反应，但与鲤血清Ig无同源性。三种鲟鱼血清IgM可被木瓜蛋白酶水解并得到两个均一的、相对分子量分别为44KD和66KD的片段，其中66KD片段免疫印迹检测结果为阳性。采用双抗体夹心ELISA法测定了这三种鲟鱼血清中IgM的平均浓度。 史氏鲟在受到嗜水气单胞菌感染的情况下，血清中补体C3、C4的含量均有所升高，其中C3的含量呈现显著性的升高，表明C3在史氏鲟抗嗜水气单胞菌感染中具有一定的作用。溶菌酶的量在史氏鲟血清和组织中总的趋势是随着温度的上升而升高，但对于不同的组织，溶菌酶在每个温度梯度之间变化的规律有所差别。血清、粘液、肝、胃及后肠在本研究的最低温和最高温时有显著性的差异，其余各组织的溶菌酶随温度变化无显著性差异。溶菌酶在各组织中分布不同，前肠的溶菌酶量在各温度下均占据优势，幽门垂、后肠及脾中溶菌酶的量也较高，而粘液和血清中溶菌酶的量较低。嗜水气单胞菌感染后的史氏鲟血清溶菌酶量呈升高趋势，但变化不显著，肝脏除在1今15℃无变仕汐卜，其余温度下较健康对照有显著性升高;史氏鳃感染后胃、鳃及粘腋熔菌酶随温度产生不同的变化;史氏鳃前肠、后肠及幽门垂在感染后溶菌酶的活性均呈现阳氏的趋势，表明不同组织在感染中的变化不同。史氏鳃与小体鳃溶菌前在组织中的分布表明，虽然同为鳃科鳃属，其差另琳王剥良显著的。 史氏瞬各组织中的碱性磷酸酶AKP周州郎明顶序为:有旗酥幽门塑乡后服卜鲤卜用卜胃:小体鳃各组织中AKP活性的排列须序为幽门夔乡蒯卧后服卜鳃明升胃。史氏鳃各组织中酸性磷酸酶ACP活性的抖陌山项序为蒯卧用卜幽门菠乡后服卜胃>鳃:小体鳃各组织中ACP活性的排列顺序为月卜幽门诬卜育旗淤后服卜鲤卜胃。史氏鳃的血清中AKP活性显著高于小体鳃，月刊胜中AKP的活性则明显低于小体鳃，两种鱼其它各组织AKP无明显差别，两种鱼各组织中的ACP活性亦无显著差异。对两个生长阶段史氏瞬及其它五种鳃鱼幼鱼胃、肠道和肝脏中蛋白酶、月哥访酶、淀粉酶周幽荆于了测定。除个另师在较大差异外，三种消化酶活性在六种鳃鱼间大体上没有明显差异。 一氧化氮NO是重要的免疫调节分子，在嗜水气单胞菌感染后的史氏鳃血清中检钡怪U了NO的显著性升高，月刊胜中的NO在感染后变化不显著。一氧化氮合酶NOS在NO的合成中起着关键的作用，但史氏鳃感染后血清及肝脏中的NOS活性无显著性的变化。 许多研究表明中药黄蔑对免疫功能具有调节作用，采用灌服的方法研究了黄茂对史氏鳃免疫功能和抗氧化能力的影响。灌服称劝口剂量为1%的黄蔑的史氏鳃与对照比肝脏溶菌酶量有显著升高，其余组织中溶菌酶量虽升高，但无统计学意义;摺貌受截茂后史氏鳃血清中NO及NOS无显著变化，但1.仓场组及2.创毛红明升吐NO的量及2.嘶组肝脏NOS活性与对照组有显著性差异，同感染试验相比，药物调节的NO及NOS变化不同;黄茂具有抗氧化能力，显著阳氏了史氏鳃血浆中的月断欣峰以户物MDA，对红细胞、血浆及肝脏SOD活性总的来说有增强作用，添加剂量为1%时效果显著。更多还原

【Abstract】 Amur sturgeon (Acipenser schrencki Brandt) is a kind of essential economic species in Heilongjiang River and now become an important culture fish. It is very useful to study the immunological factors and other related factors of the fish for curing and preventing diseases and adopting immunization techniques in the fish culture.Blood cells are important in immune system. Reticulocyte and erytnrocyte which are splitting were found There are four kind of leukocytes in peripheral blood of Amur sturgeon: lymphocyte, granulocyte, tnrombocyte and monocyte. Blood cells of the fish were observed under electron microscope. There are a little organells in the cytoplasm of the erythrocyte. The lymphocyte is typical and there are lots of mitochondria in cytoplasm of the monocyte which is smaller than granulocyte. Thrombocyte is in fusiform or nearly round form with little cytoplasm. The granules of granulocyte are classified into two kinds based on electron-lucent and electron-dense.Serum protein and Ig of 2+ years old Amur sturgeon, 3+ years old Chinese sturgeon Acipenser sinesis Gray and 3 years old Huso sturgeon Huso dauricus Georgi are analyzed. The results show that serum proteins of the three fishes are different with each other in composition and level of serum proteins. Coefficient of similarity about serum protein show that Chinese sturgeon is more similar with Amur sturgeon compared with Huso sturgeon. Saturation ammonium sulfate sedimentation and Sephadex G-200 gel chromatography were used to purify Ig from serum of Amur sturgeon, Chinese sturgeon and Huso sturgeon respectively. IgM molecular weight of Amur sturgeon, Chinese sturgeon and Huso sturgeon are 867KD, 896KD and 924KD respectively assayed by unreduced PAGE. Reduced SDS-PAGE shows that heavy chain molecule weight of IgM in the three Sturgeons serum are all 88KD and light chain molecule weight of Amur sturgeon and Chinese sturgeon are 29KD, while there are two kinds of light chain in Huso sturgeon with molecule weight at 29KD and 26KD. Western-blotting analysis showed antibody activity of IgM and the heavy chain of IgM in serum of die three Sturgeons. Ouchterlony results show that IgM of each of the three Sturgeons can precipitated with rabbit serum anti IgM of each other, but cannot response with rabbit serum anti carp IgM. IgM of the three Sturgeons can be hydrolyzed by papain in corresponding conditions and two fragments are obtained. Molecule weight of first peak is 66KD and that of the second is 44 KD determined by SDS-PAGE. Content of IgM in sera of Ihe three Sturgeons were assayed by sandwich ELISA with sensitivity at 16ng.ml-1.C3 content in serum increased significantly while the fish infected by Aeromonas hydrophila (Ah) and C4 showed insignificant in the same condition It seemed that C3 is important in Ahinfection. Lysozyme content in serum and other tissues of 1+ years old Amur sturgeon tended to increase with the temperature, but increasing range varied in different tissues. Lysozyme content in serum, mucus, liver, stomach and hindgut showed significant difference between the lowest and the highest temperature in the test while that in the other tissues showed no significant difference. We analyzed distribution of lysozyme in serum and several tissues of Amur sturgeon and the results show that lysozyme content in foregut is the highest in the tissues assayed in every temperature in the test while lysozyme in pylonc caecum, hindgut and spleen show hight level in the test and that in serum and mucus are lower in level compared with the other tissues. Lysozyme content in serum increased insignificantly after Amur sturgeon infected by Ah compared with that in the normal fish. Lysozyme in liver showed significant increasing between normal fish and fish infected except at 14-15℃. Difference of lysozyme in stomach, mucus and gill between nonnal fish and fish infected varied in different temperature. Lysozyme in foregut, hindgut and pylonc caecum decreased after the fish infected by Ah. The changes of different tissues were not the same after更多还原