【作者】 刘云；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2003， 博士

【摘要】 近年来，鱼类免疫系统的形态和功能的研究日益重视，一方面，从系统进化角度考虑，鱼类同其他脊椎动物一样，也具有有效的免疫防御系统，有关鱼类免疫系统的研究，将有助于阐明人类免疫系统的起源和进化；另一方面，随着世界人口的增长和对水产品消费需求的提高，硬骨鱼的高密度养殖引起了水产病害的广泛传播，如大量使用抗生素会影响人类食品安全，而利用疫苗防治疾病具有明显的优越性。因此，对鱼类免疫系统的研究，不仅可以研究脊椎动物免疫系统的进化规律，而且对鱼类的健康养殖和持续发展有着重要的应用价值。 牙鲆是我国北方人工养殖的重要的海水经济鱼类。本文以牙鲆为代表，首次全面系统地研究了海水鱼类——牙鲆的免疫系统，包括头肾、脾脏、胸腺，血液以及粘膜样淋巴组织的组织结构和细胞特征。首次全面系统地研究了海水鱼类——牙鲆免疫系统发生及其1个月到第13个月的发育变化，阐明了免疫器官发育与年龄、体重等关系，同时发现了牙鲆胸腺快速退化现象。通过对牙鲆特异性免疫球蛋白进行的分离纯化得到牙鲆免疫球蛋白，其重链分子量75.4KD，2条轻链分子量分别为29.9KD和28.2KD。同时，还对鱼类免疫系统与环境的关系，特别是病原菌感染与免疫系统的结构和功能的影响进行了进一步的研究和分析，指出血液中白细胞数目和生化成分的变化可以作为鱼病早期诊断的良好指标。 牙鲆的免疫器官包括肾脏(头肾)、胸腺、脾脏、血液和粘膜样淋巴组织包括鳃与肠。牙鲆的头肾主要集中于肾脏前部2个分叉部分；组织学研究证明，头肾是由基质组织网络着不同发育阶段的血细胞构成，无肾小球等分泌结构；超微观察进一步表明，基质是由网状细胞和成纤维细胞等组成，各种免疫细胞由淋巴细胞、单核细胞、Ⅰ型颗粒细胞和Ⅱ型颗粒细胞以及巨噬细胞等组成。牙鲆头肾内免疫细胞种类比较丰富，是免疫细胞的主要生发中心。牙鲆脾脏实质无明显的红髓和白髓区分。胸腺无明显的内区和外区之分。 牙鲆外周血液中包括六种类型的血细胞：红细胞、淋巴细胞、血栓细胞、单核细胞、嗜中性颗粒细胞和嗜酸性颗粒细胞，未见嗜碱性颗粒细胞。淋巴细胞的体积最小，单核细胞的体积最大；在白细胞中，淋巴细胞数量最多，嗜酸性粒细胞数量最少；此外，还可见到正在分裂的血栓细胞，说明血栓细胞可在外周血液中经直接分裂产生。电镜研究结果表明，牙鲜红细胞的细胞质均匀，有少量线粒体、囊泡和大量的极细颗粒；淋巴细胞的细胞核大且中位，细胞质中含较多的游离核糖体，胞体具有短伪足；单核细胞的细胞核偏位且形态不规则，细胞质中有内吞泡和较多的细胞器；颗粒细胞可根据颗粒的不同形态分为1型和＊型两种类 二型。 牙鲜的鳃小片主要由扁平上皮细胞和柱细胞构成，血窦腔极为发达。鳃小片在功能上可分为两个区：气体交换区和免疫区。气体交换区位于上半部分，血窦内主要分布着红细胞；免疫区位于鳃小片基部，血窦腔中分布着各种兔疫相关细胞：淋巴细胞、单核细胞、嗜中性颗粒细胞和嗜酸性颗粒细胞。此外，还观察到了泌氯细胞和粘液细胞。研究结果证明，牙鲜的鳃在局部粘膜免疫中具有重要的作用，是鱼类免疫体系当中的有机组成部分之一。 在牙鲜肠淋巴样组织中鉴定出5种白细胞，即淋巴细胞、巨噬细胞、单核细胞、浆细胞样细胞和颗粒细胞。它们的形态均比在造血器官中多样化。淋巴细胞具有多且细长的伪足；巨噬细胞形态极其不规则；单核细胞的细胞质中有较多空泡；而浆细胞样细胞是以内含大量的粗面内质网为其主要特征。牙鲜肠淋巴样组织内颗粒细胞经鉴定为嗜酸性颗粒细胞，常位于肠粘膜层及粘膜下层靠近肌肉层的淋巴腔中，以具有大型非匀质颗粒为其主要特征，其变化可分为增长期、成熟期、分泌期和衰退期四个时期。同样，肠淋巴样组织也是鱼类免疫防御系统的重要组成部分。 从牙稣孵化到第40天，系统的研究了牙鲜免疫器官的发生。头肾在孵化时即已经出现，7天后出现造血干细胞；脾脏在第2阶段即第3～9天时出现；胸腺在第3阶段末期出现，位置靠近头肾。小淋巴细胞出现的顺序是胸腺、肾脏和脾脏。非特异性防御体系在牙稣早期发生中起了重要的作用。 从1个月到13个月，系统地研究了牙解免疫器官的发育情况。除了胸腺之外，所有免疫器官都随着鱼类年龄的增长而增长。2个月时免疫器官达到相对的最大值。免疫器官的重量与体重的关系要比与年龄的关系密切。免疫器官内总的白细胞数量随着年龄增长而增长，但考虑到免疫器官的重量，实际的白细胞数量基本 恒定不变。显微和超微结构研究都表明，牙稣免疫器官发育直到幼鱼阶段才逐步 完善。脾脏和头肾有混合的红细胞和白细胞群，头肾比脾脏更加淋巴化。胸腺在 孵化后前6个月快速退化。血液组成与年龄或季节的关系不明显，红细胞占绝对 优势。与其它鱼类相比，牙鲜淋巴器官的发育比较晚，牙稣的淋巴器官不完善。 所有这些都表明牙鲜的免疫系统淋巴化程度不高，可能会被多种病害侵害。一 在?更多还原

【Abstract】 In recent years the information regarding the morphological and organization of fish immune systems has increased dramatically. Study on the fish immune systems will not only contributes to the understanding of the origin and evolution of human immune system, but also to the farming of the fish.Flounder, Pamlichthys olivaceus, was an important species in the aquaculture in China. This paper studied the structure of the immune system, including head kidney, thymus, spleen, blood and mucosa-associated lymphoid tissues, such as gill and gut for the first time. In addition, the ontogeny of the immune system in the flounder from hatching to 13 month was also studied. The thymus involuted quickly than any other fishes. The immunoglobulin was purified from serum of the flounder. It was found that the heavy chain was 78kd, and the two light chains were 29.9kd and 28.2kd. The ultrastructural and biochemical study of the flounder infected with Edwardsiella tarda were also studied. The changes in the leucocytes number and the biochemical parameters were determined, and they could serve as candidates of biomarkers for surveillance of infection of the flounder.The immune system of the flounder included kidney (head kidney), thymus, spleen, blood and mucosa-associated lymphoid tissues, gill and gut et al. Head kidney of the flounder was restricted in the foremost parts of kidney, which was predominantly a lympho-myeloid compartment with aglomerular. The parenchyma of head kidney was supported by the stroma of the kidney. The stroma consisted of reticular cells and fibroblasts. The cells responsible for the nonspecific and specific immune defenses included lymphocytes, monocytes, granulocytes (two types) and macrophages. The results showed that the immune cells in head kidney were abundant and head kidney might be an important germinal center. There had no distinct inner or outer zones in thymus and red or white pulp in spleen.The micro- and ultrastructure of peripheral blood cells of the flounder were studied. Under the light microscopy, six main types of blood cells could be recognized: erythrocytes, monocytes, thrombocytes, lymphocytes, neutrophils and eosinophils. Basophils were not found. Among these cells, lymphocytes were the smallest, while monocyteswere the biggest; Lymphocytes were the most abundant leucocytes and eosinophils were the least. The direct division of thrombocytes could be formed in the peripheral blood. The transmission electron microscopy showed that cytoplasm of erythrocytes was even and only has a few mitochondria, vacuoles and many small black granules. Lymphocytes were characterized by the presence of short pesudopodia, the big center nucleus and many free ribosornes, while monocytes were characterized by eccentric nucleus, vacuoles and many organelles. Two distinctive types of granules leucocytes were also described as type I and type II.The gill lamella was composed of squamous epithelium cells and pillar cells. The blood sinusoid cavity was very large. Functionally, the gill lamella could be divided into gas exchange region and immune region. Gas exchange region located in the upper half with many erythrocytes in the blood sinusoid, while the immune region located in the lower half with lymphocytes, monocytes, neutrophils and eosinophils in the blood sinusoid. Moreover, the chloride secreting cells and mucous cells were also observed. The results indicated that the immune cell types existed in the gill tissue was completed and they might play some extent roles in the fish local immune competence.Five kinds of leucocytes in the gut-associated lymphoid tissue (GALT) of the flounder were distinguished under the transmission electron microscope. They were lymphocytes, macrophages, monocytes, plasma-like cells and granulocytes. The leucocytes in the GALT all showed more diversified than that in the hematogenic organs. Lymphocytes were identified by many long pseudopodia. Macrophages had irregular shapes. Many vacuolus occurred in the monocytes’ cytoplasm and plasma-like cells wer更多还原