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

肿瘤坏死因子是免疫调节和炎症反应的主要介质。肿瘤坏死因子也被认为是参与内分泌、神经和免疫系统相互作用的媒介。也有研究报道，肿瘤坏死因子/肿瘤坏死因子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，肿瘤坏死因子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。为了了解肿瘤坏死因子广泛的生物活性，我们试图从系统发生学和个体发生学的角度研究其多效性。在本研究中，我们获得了以下结果：1.鱼类肿瘤坏死因子的结构与功能我们从金枪鱼中分离到一个肿瘤坏死因子同源物，从虹鱼和斑马鱼中分离到两种肿瘤坏死因子同源物。我们还从软骨鱼中克隆了肿瘤坏死因子诱骗受体3(DcR3)和几种细胞因子。2.重组肿瘤坏死因子的产生及其生物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产物3.细菌和/或内毒素刺激后组织中肿瘤坏死因子的表达不同于两种类型的肿瘤坏死因子在虹鱼体内的表达。利用与绿色荧光蛋白(GFP)连锁的肿瘤坏死因子启动子(GFP)为报告基因的转基因虹鱼的研究表明，肿瘤坏死因子分泌细胞参与了对细菌的包膜反应。4.肿瘤坏死因子在发育中的作用在斑马鱼胚胎的神经系统中有两种类型的肿瘤坏死因子弱表达，而在阿育胚胎中观察到在孵化过程中肿瘤坏死因子的表达增加，提示肿瘤坏死因子在胚胎器官的形态发生中起作用。

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.