【作者】 王文君；

【导师】 张志峰；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2008， 硕士

【摘要】 鱼类肝脏和脾脏是鱼类生物学研究的重点对象。肝脏是鱼类维持体内代谢平衡的重要器官,能调节营养平衡,合成和分泌多种代谢物质。脾脏是一个多功能复合器官,在鱼类的造血和免疫中有重要作用。大泷六线鱼具有较高的经济价值,是近年来新兴的一种海水鱼类养殖品种,然而其生物学背景资料较少。本文采用组织学和细胞学方法对大泷六线鱼的肝脏和脾脏进行了结构研究,进一步对其细胞的体外原代培养进行了研究,旨在丰富大泷六线鱼的生物学研究内涵,为其细胞系的建立奠定基础。组织学和细胞学研究表明,大泷六线鱼肝脏内结缔组织不发达,肝小叶不明显,中央静脉不规则,肝细胞索围绕中央静脉呈不规则放射状排列,肝血窦狭窄。肝门管区不典型。肝细胞较大,呈多边形,细胞间分界明显,细胞质丰富,细胞核大,多为圆形或椭圆形。脾脏被膜较薄,未见明显脾小梁,红髓和白髓混合分布,界限不清,构成脾脏的细胞种类包括淋巴细胞、红血细胞、粒细胞、网状细胞、巨噬细胞等,有大量黑色素巨噬细胞中心分布。本文细胞培养的实验目的是为了建立和优化大泷六线鱼细胞体外培养的体系。在肝脏的原代培养中,通过正交实验和随后进行的两因子三水平实验,分别对培养基种类、血清浓度以及渗透压水平进行了筛选,结果表明,在不改变培养基原有渗透压的条件下,使用MEM培养基,添加15%胎牛血清可以取得较好的实验结果。在该培养条件下,细胞迁出旺盛,迁出细胞呈椭圆或多角形,可存活14d左右。在脾脏原代培养实验中,使用MEM培养基的培养效果明显好于L-15培养基,在10%、15%、20%的血清梯度下,都可以长期维持脾脏细胞的体外培养。其中,C型细胞(成纤维样细胞)在三种血清浓度下均可生长,以含15%胎牛血清的培养基中生长最好,至论文完成前已培养近4个月,目前细胞生长状况正常;D型细胞(巨噬样细胞)则在含20%胎牛血清的培养基中生存时间最长,培养50d时,仍然正常生存。此外,在脾脏细胞原代培养中观察到C型(成纤维样细胞)细胞层上出现了造血灶,这些位置可持续产生圆形、明亮的细胞并将其释放到培养液中,因此推测C型细胞是某种可以支持造血活动的基质细胞,而大泷六线鱼的脾脏也具有活跃的造血功能。更多还原

【Abstract】 Liver is considered to be an important organ due to its functions in maintaining the internal homeostasis in fish. The spleen of fish is believed to be involved in haematopoiesis, the clearance of macromolecules, antigen degradation and processing and the production of antibody. The liver cultures and spleen cultures from fish have been proved to be good models for the study of metabolism and physiology as well as for the research of toxicology and carcinogenesis in vitro.Hexagrammos otakii is an important commercial marine teleost fish and its aquaculture has being developed in costar area of northern China since recent years. However, the research on its basic biology is little by now.In this paper, the micro-structure and ultra-strcture of liver and spleen in Hexagrammos otakii were studied by histological and cytological technique. The primary culture technique for the splenic cells and hepatic cells from Hexagrammos otakii is described for the first time. The conditions for primary culture have been determined.The results from liver histology research of H. otakii showed that the connective tissue was less and the hepatic lobule was not distinct. Central vein was irregular, the hepatic cord was radial around central vein, and hepatic sinusoid was irregular. Portal area was not typical. Boundaries of hepatocytes were clear, the hepatocyte was large and polygonal, cytoplasm was plentiful. The results of spleen histology research indicated that the splenic capsule was very thin and the trabeculae were poorly developed. The chief characteristics of the spleen were the pulp and melano-macrophage centers. The spleen parenchyma was composed of white pulp and red pulp, but there was no sharp demarcation between them. The major cellular components were erythrocytes, lymphocytes, reticular cells, granulocytes and macrophages. The melano-macrophage centers were usually located near vessels, but the masses of melano-macrophage were not surrounded by circum adjacent cell layers.The purpose of cell culture study in this paper is to explore proper methods and culture conditions for the initiation of cell cultures from liver and spleen in H. otakii.In order to find out and optimite the culture conditions for the hepatic cells, the orthogonal design experiments which took culture medium (L-15,DMEM,MEM), serum concentration (10%,15%,20%), osmotic pressure (normal NaCl concentration, 10g/L, 15g/L) as the factors and furthermore two-factor three-level experiments which took serum concentration (10%,15%,20%), osmotic pressure (normal NaCl concentration, 10g/L, 15g/L) as the factors were conducted. The results showed that the proper culture condition of hepatic cells was MEM with 15% fetal bovine serum (FBS) without additional NaCl in the medium. Under this culture condition, the cultured cells were highly homogeneous, they grew well and could survive for 14 d or so.During the primary culture of splenic cells from H. otakii, the culture medium (L-15, MEM), serum concentration (10%, 15%, 20%) were also been chosen to screen the culture condition. The results indicated that MEM was the better medium to initiate the culture of splenic cells from H. otakii. Different FBS concentrations supported different types of cells growing. Type C cells (fibroblast-like cells) could grow into monolayer or multilayer in MEM with all FBS concentrations, but they grew best in MEM with 15% FBS. Under this culture condition, Type C cells could outgrow from explants and cover approcimately 80% of the growth surface which was the most among three FBS concentrations. The optimum culture condition for the growth of Type D cells (macrophage-like cells) was MEM with 20% FBS in which the cells could survive longer (50d) than the other two conditions.The hematopoietic foci were observed after the first change of medium in the spleen cultures. The foci were formed by round phase bright cells loosely attached on top of type C cells (fibroblast-like cells), some of which were subsequently released into the medium. It was inferred that type C cells maybe formed a stromal cell layer which played a role in haematopoiesis and the spleen of H. otakii was an important haemopoietic organ as in other teleosts.更多还原