Studies on the structure and function of tumor necrosis factor (TNF) in fish

肿瘤坏死因子（TNF）是免疫调节和炎症反应的主要介质。TNF也被认为是参与内分泌神经和免疫系统之间相互作用的介质。TNF/LT家族细胞因子在次级淋巴器官的发育、成熟和维持中起重要作用。因此，TNF是迄今为止从各个领域和方面研究的最重要的细胞因子之一。为了理解TNF的广泛的生物活性，我们试图从胚胎发生学和个体发生学的观点来研究其多效性。1.鱼类TNF的结构与功能我们从香鱼中分离到一种TNF同源物，从虹鳟鱼和斑马鱼中分离到两种TNF。我们还从软骨鱼中克隆了TNF诱饵受体3（DcR 3）和几种细胞因子。重组肿瘤坏死因子的产生及其生物学活性纯化的重组肿瘤坏死因子可诱导头肾白细胞产生超氧化物歧化酶。3.脂多糖和/或细菌刺激后虹鳟鱼TNF mRNA的表达脂多糖刺激后两种TNF在虹鳟鱼组织中的表达存在差异。以绿色荧光蛋白（GFP）为报告基因的转基因虹鳟鱼为研究对象，研究表明，TNF分泌细胞参与了对细菌的免疫应答。4. TNF在发育中的作用两种类型的TNF在斑马鱼胚胎神经系统中弱表达，TNF在胚胎发育过程中的表达量随时间的延长而增加，提示TNF在胚胎器官的形态发生中起重要作用。

Tumor necrosis factor (TNF) is a primary mediator of immune regulation and the inflammatory response. TNF is also known to be a mediator involved in the interaction between endocrine nervous and immune systems. There have been also reported that TNF/LT family cytokines play important role in development, maturation and maintenance of secondary lymphoid organs. Thus TNF is one of the most important cytokines investigated so far from various fields and aspects.To understand the broad range of biological activities of TNF we tried to investigate its pleiotropic nature from phylogenetical and ontogenetical points of view. In the present research we obtained the following results :1.Structure and function of fish TNFWe isolated one TNF homologue from Ayu fish and two types of TNF from rainbow trout and zebrafish. We also cloned TNF decoy receptor 3 (DcR3) together with several cytokines from cartilaginous fish. Structure and expression of these fish TNF and TNF super family genes resembles to those of mammalian TNF.2.Production of recombinant TNF and its biological activityPurified recombinant TNF of Ayu fish induced the production of superoxide from head-kidney leukocytes.3.Expression of TNF mRNA after stimulation with LPS and/or bacteriaExpression of TNF in tissues after stimulation with LPS was different between two types of TNF in rainbow trout. Studies using transgenic rainbow trout introduced with TNF promoter linked to green fluorescence protein (GFP) as a reporter gene showed that TNF secreting cells are involved in envelopment response against bacteria.4.Role of TNF in developmentTwo types of TNF were weakly expressed at the nervous system of zebrafish embryos, and increased TNF expression toward hatching was observed in Ayu embryos suggesting the role of TNF in the morphogenesis of embryonic organs.