每年病害都会给鱼类养殖造成重大的经济损失，因此鱼类养殖上采用了包括遗传育种、疫苗免疫以及使用免疫增强剂等一系列措施来减小因病害造成的损失，而这些措施的合理应用有赖于对鱼类免疫系统的深入了解。目前，普遍认为鱼类具有CD4+ 和CD8+ T细胞，但尚不清楚CD4+ T细胞是否可以如哺乳动物的一样可以进一步分为辅助性T细胞1型（Th1）、2型（Th2）、17型（Th17）和调节性T细胞（Treg）等细胞类型。在前面的工作中，本实验室已经克隆出大西洋鲑（Salmo salar）GATA3（Th2细胞关键性转录因子）和IL-17D（Th17特征性细胞因子）。在此基础上，本研究又克隆了T-bet（Th1细胞关键性转录因子）和Foxp3（Treg细胞关键性转录因子）两个基因 ，并进一步研究了不同类型的CD4+ T细胞因子在抗病品系选育及施以免疫增强剂情况下的组织表达情况，得出的主要结论如下：1. 大西洋鲑T-bet基因cDNA全长2293bp, 其中开放阅读框长1839 bp, 编码的蛋白含612个氨基酸。其具有一个保守的T-box DNA结合结构域。通过与其它鱼类及哺乳动物T-bet基因的分析，发现大西洋鲑T-bet基因的基因位置较为保守。蛋白水平的进化树分析也显示大西洋鲑的T-bet与其它动物的T-bet聚在一起，与Tbr1和Eomes这两个T-box 亚家族成员形成明显不同的三个聚类，表明鲑鱼的T-bet基因与哺乳动物的具有同源性。实时荧光定量PCR检测发现其在胸腺的表达量最高。在大西洋鲑头肾原代细胞培养实验中，相对于对照组，经ConA（T细胞特异性丝裂原）刺激后T-bet基因表达量随着时间先升高后略有降低；经β-葡聚糖刺激后，T-bet基因表达量也是随时间先升高后降低；而经LPS（B细胞特异性丝裂原）刺激后T-bet基因表达量与对照组无异，且不随时间发生改变。由于个体间差异较大，三者的变化均不显著（ConA, F2,17=0.737, p=0.628）。经杀鲑气单胞菌（Aeromonas salmonicida）活菌腹腔注射的大西洋鲑，脾脏T-bet基因表达未发现明显变化，而头肾的T-bet基因表达量在48小时内逐渐升高。因此，大西洋鲑T-bet基因有可能与其同源的哺乳动物T-bet基因具有相类似的功能。2. 大西洋鲑Foxp3基因cDNA全长1865bp, 其中开放阅读框长1332bp, 负责编码一个长为443个氨基酸的蛋白质。软件预测其蛋白与哺乳动物的Foxp3一致，含有一个Forkhead结构域，一个leucine-zipper 结构域和一个C2H2锌指结构域。蛋白水平的进化树分析也显示，其与其它动物的Foxp3聚在一起，并与Foxp1、Foxp2和Foxp4分别形成明显的四个聚类，这表明Foxp3基因比较保守且同源性很高。Foxp3基因插入含cmv 启动子的RFP质粒后转染CHSE-214细胞，发现红色荧光集中在核内和核周围，而对照组（空质粒）的红色荧光散在细胞质中。这表明大西洋鲑Foxp3基因是核定位的基因。组织分布检测发现，大西洋鲑胸腺中Foxp3 mRNA的丰度最高。大西洋鲑头肾原代细胞培养实验中，与对照相比，经ConA刺激的头肾细胞Foxp3基因在刺激24小时后表达逐渐降低；而β-葡聚糖刺激24小时Foxp3基因表达量增加，在48小时表达量出现降低；而LPS刺激的头肾细胞在24小时后 Foxp3基因表达量逐渐增加。经杀鲑气单胞菌活菌腹腔注射的大西洋鲑，脾脏Foxp3 mRNA水平先升高后恢复到正常水平，而头肾Foxp3 mRNA水平呈现U型变化，攻毒48小时达到最高。结果表明大西洋鲑Foxp3基因很可能具有其哺乳动物同源基因相类似的免疫抑制功能。3. 两个具有不同抗病力的大西洋鲑品系（耐受品系和易感品系）经自然途径感染杀鲑气单胞菌后，总的来说，脾脏和头肾中的两种炎性反应指标IL-1β和TNF-α基因表达量、鞭毛蛋白受体TLR5的膜型和分泌型的mRNA表达量均显著增加，且耐受品系高于易感品系，这表明机体发生了炎性反应。而在肝脏中，耐受品系与易感品系中上述基因的表达趋势正好相反。CD4+ T细胞免疫因子GATA3、IFN-γ、IL-17D和抑制炎性的细胞因子IL-10在头肾和脾脏中的基因表达量也显著增加，且耐受品系增加量高于易感品系，而在肝脏中，趋势各异。与此不同的是攻毒前耐受品系补体溶血活性显著高于易感品系，而攻毒后两个品系间没有显著差异。这些结果表明，上述提到的免疫因子可以作为潜在的定向育种选择标志。4. 虹鳟（Oncorhynchus mykiss）幼鱼分别浸泡两个不同剂量（0.1 μM 和 1.0 μM）的三种免疫增强剂（质粒DNA、乳铁蛋白和β-葡聚糖）后，结果显示，与对照组相比，质粒DNA处理组、乳铁蛋白处理组以及β-葡聚糖处理组在第一次浸泡后的三个取样时间点，转录因子T-bet（F5,143=12.855, P < 0.001）、促炎性细胞因子IL-1β （F5,141=7.231, P < 0.001）、TNF-α（F5,139 =17.192, P < 0.001）、IL-6（F5,139 = 13.334, P =0.033）及抗炎性细胞因子IL-10（(F5,139=13.334, P < 0.001）和TGF-β （F5,144 =17.192, P < 0.001）与对照组相比，转录水平显著上升，这一点在高剂量组中尤其明显；而最后一次浸泡后的两个取样时间点，与对照组相比，上述细胞因子转录水平未见显著变化。各处理组促炎性细胞因子IL-17A转录水平在各时间点均未见到明显变化（F5,102 =1.563，P = 0.177）。这些结果表明虹鳟幼鱼多次浸泡免疫增强剂可能导致免疫耐受，在浸泡后还可能存在Th1型免疫反应，Treg细胞的细胞因子在浸泡后的显著上升可能表示伴随免疫细胞的负调控。本研究结果进一步提示鱼类CD4+ T细胞存在可以分为不同亚型的可能，为深入研究鱼类是否存在CD4+ T细胞亚型提供了基础，也为鲑鳟鱼类的抗病力定向遗传育种和免疫增强剂的合理应用提供一定帮助。

Diseases cause big economic losses in fish production each year, and many measures have been developed, such as selective breeding, immunostimulants and vaccines, and applied to counteract these losses. However, currently most of these applications, in particular vaccination, has not been fully satisfactory enough to attenuate the losses, which may be recognized as being due to the lack of a rational vaccine design. It is acknowledged that the development of fish immunology may contribute to the solution of this problem. In the present study we focus on cytokines and transcription factors of salmonid fish where the mammalian counterparts have been shown to be critical for CD4+ T cell differentiation, in an attempt to decipher T cell lineages in salmonid fish. The salmon (Salmo salar) T-bet gene was cloned and characterized in this study. Sequence analysis revealed that it contained a conserved T-box DNA-binding domain, which is a typical characteristic of the mammalian T-bet gene. Gene synteny and phylogenetic analysis indicated it was a homologue to T-bet gene in mammals. Following live bacteria challenge in vivo, and mitogens and β-glucan stimulation in vitro, the expression pattern of salmon T-bet further suggested it to be an orthologue of mammalian T-bet.The cloning and molecular analysis of salmon Foxp3 revealed that, as its mammalian counterpart, it possesses a Forkhead domain, a leucine-zipper domain as well as a C2H2 zinc-finger domain. Phylogenetic analysis further suggested that it belonged to the Foxp3 cluster, distinct from Foxp1, Foxp2 and Foxp4 clusters. The transfection of salmon Foxp3 tailed with a RFP in CHSE-214 cells clearly demonstrated that it is a nucleus-located molecule. The regulated Foxp3 mRNA expression pattern after stimulation in vitro and in vivo indicated that salmon Foxp3 has evolutionarily conserved functions.Expression pattern of immune molecules (IL-1β, TNF-α, TLR5M, TLR5S, GATA3, IFN-γ, IL-17D and IL-10) from two Atlantic salmon families with different defense ability against Aeromonas salmonicida infection suggested that some of the genes might be potentially indirect markers of disease resistance in selective breeding. However, it was difficult to conclude whether, if any, a Th1, Th2 or a Th17 type immune response in Atlantic salmon was induced by the challenge of A. salmonicida.In the experiment where rainbow trout (Oncorhynchus mykiss) fry were bathed with immunostimulants (plasmid DNA, lactoferrin, β-glucan) at two different doses (0.1 μM and 1.0 μM), a regulated expression of the cytokines and transcription factors, whose mammalian homologues are critical for CD4+ T cell development, was observed, and the expression pattern implied that there might be a Th1 type immune response. Furthermore, repeated bathing did not boost this response, but seemed to induce an immune silence. All together, the results suggest that it is likely that fish possess different subtypes of T helper cells as mammals do, and they are likely to have conserved function in the response to immunostimulants. These critical cytokines as well as the key transcription factors could be potentially applied as indirect selection markers in fish breeding. The current study particularly facilitates the searches for the presence of different subtype CD4+ T cells in salmonid fish by means of gene characterization and immune stimulation.