鱼类的自体抗病能力

所有动物为了维持它的正常生理功能，都必须具备各种各样的防止病原体侵入体内的防御构造和功能。从系统发育的观点来看属低等脊柱动物的鱼类，为了在水这个特殊环境中生息，它的生理特性与其他动物在自体抗病的结构和功能上有区别。一般而言，鱼类机体能识别非自身的病原体的侵入，初期阶段鱼类起自体抗病力作用的是非特异性防御系统，后期主要是特异性免疫系统对病原进行防御。本文对鱼类自体抗病能力有关的特异性和非特异性的组织器官的作用简述如下。一、非特异性防御系统１．粘液在鱼体表的表皮层存在着杯状细胞。杯状细胞分泌出粘液覆…     

鱼类的干扰素系统及干扰素功能

干扰素系统是机体对抗病毒感染的一道重要防御系统,它与细胞免疫、体液免疫及其他非特异性免疫协同作用抵抗病毒的侵染。在干扰素系统中产生的干扰素是一种广谱抗病毒剂,其作用是通过对细胞表面受体作用使细胞产生抗病毒蛋白,抑制病毒在宿主细胞中的复制。还可增强自然杀伤细胞（NK细胞）、巨噬细胞和T淋巴细胞的活力,从而起到免疫调节作用,增强抗病毒能力,在一定程度上还影响细胞生长和分化等多种生物活性。本文将对鱼类的干扰素系统及干扰素功能作综述报道。     

鱼类树突状细胞研究进展

树突状细胞(DCs)是目前已知的体内功能最强的抗原递呈细胞,是唯一能够激活初始T淋巴细胞反应的细胞,在先天性免疫、适应性免疫以及维持自身免疫耐受方面具有重要的作用,因此一直是免疫学研究的重要领域。本文简要综述了DCs的类型及其在动物体内的功能、各类DCs的细胞标记。总结了鱼类DCs的分离、纯化方法和形态学观察方法;现有研究表明,鱼类DCs具有吞噬细菌、刺激T细胞增殖、诱导CD4+T细胞的活化、表达DCs的标记基因、被Toll样受体的配体激活、迁移能力、引起混合淋巴细胞反应等生物学功能;不同鱼类DCs的分子标记并不完全一样;鱼类的头肾、肾、鳃、皮肤、胸腺、脾、肠等均有DCs的分布。目前,对鱼类DCs的研究虽然取得了一定进展,但仍有许多重要问题需要解决:①鱼类DCs目前缺乏明确的细胞标记,加强这方面的研究有助于提高鱼类DCs的分离、体内分布与功能的研究水平;②加强和完善鱼类DCs的分离、培养技术的研究,掌握各种鱼类DCs的分离培养方法;③加强鱼类DCs在抗原递呈中的功能研究,对深入分析鱼类免疫机理,合理设计和应用疫苗,具有重要的理论指导意义。     

鱼类淋巴细胞异质性的研究概况

鱼类抗病的免疫功能,很大程度上取决于免疫细胞的作用。免疫细胞由非特异性的吞噬细胞、特异性的淋巴细胞以及天然杀伤细胞构成(张玲等,1997）。所有免疫细胞的共同特点之—是功能与形态上的多样性,因此,对免疫细胞进行分类后在体外获得代表性亚群进行研究,是认识各类免疫细胞功能和免疫反应机制的重要条件。     

鱼类胃肠道内分泌细胞的研究进展

免疫组织化学技术是近年来迅速发展起来的一门新的边缘学科 ,它具有灵敏度高、特异性强、定位准确和应用广泛等优点 ,能够对胃肠道内分泌细胞进行准确地鉴别和定位[1] 。该技术的应用为鱼类胃肠道内分泌细胞的研究开辟了有效途径 ;同时对鱼类胃肠道内分泌细胞的不断研究也推动了免疫组织化学技术的进一步发展。迄今为止 ,国内外学者对鱼类胃肠道内分泌细胞的鉴别和定位已作了大量工作。1　鱼类胃肠道内分泌细胞的种类对鱼类胃肠道内分泌细胞进行鉴别和定位均使用哺乳动物的抗血清 ,迄今为止还未发现使用专门的鱼类抗血清对鱼类胃肠道内分泌…     

鱼类补体系统的研究进展

鱼类补体大约由30余种蛋白裂解酶、酶抑制因子和受体构成的,是机体内最为复杂的限制性蛋白溶解系统。在鱼类免疫系统进化中,补体的出现比免疫球蛋白还要早。许多研究表明鱼类补体直接参与机体防御,其生物学活性影响机体抵抗微生物的能力、免疫反应细胞间的通讯联系、免疫复合物的形成和持续时间等。     

鱼类的非特异性免疫概述

对于鱼类免疫的研究很早就出现,鱼类是特异性免疫和非特异性免疫并存的脊椎动物。但与哺乳类相比,鱼类的特异免疫系统尚不够发达,特异性免疫机制还不完善。因此,在鱼类对外界刺激及病原生物侵袭的防御反应中,非特异性免疫起着重要作用。现已知参与鱼类非特异性免疫反应的因子主要包括巨噬细胞、粒细胞和细胞毒性细胞等一些具有吞噬作用的细胞,以及介导一系列免疫或应激反应的蛋白分子,如补体、细胞因子、趋化因子及溶菌酶和抗菌肽等。     

鱼类免疫系统的研究进展

<正>与哺乳动物不同,鱼类属于变温、低等动物。鱼类免疫系统主要由免疫组织与器官、免疫细胞和免疫因子组成,通常是指机体执行免疫应答和免疫功能的组织系统,是机体自身识别"自我"与消除"非我",进而排除异己的一个功能体系。其主要功能有免疫防御、免疫监视及免疫自稳三方面。鱼类的免疫系统知识仍然在不断的完善过程中,与哺乳类一致,鱼类免疫也可分为特异性和非特异性免疫两大部分。一般来说,非特异性免疫     

介导鱼类孕激素快速、非基因作用的受体研究进展

许多研究发现某些孕激素受体介导的生物学反应较迅速,而经典的通过基因起作用的孕激素核受体介导的生物学反应都较慢,因此有学者提出生物体可能存在不通过基因起作用的孕激素受体,并研究了其结构和功能,探讨了其快速反应的机制。迄今为止,在鱼类已发现了3类可能的非基因作用的膜受体。第1类为孕激素脂联素受体家族(PAQR)的3个成员,即PAQR7(mPRα),PAQR8(mPRβ)和PAQR5(mPRγ),这些成员存在于多种硬骨鱼类中,具有和G蛋白偶联受体(GPCRs)相似的7次跨膜结构域。其中对mPRα和mPRβ的研究较多,它们可以诱导鱼类卵母细胞成熟和增强鱼类精子的活动性。第2类为孕酮受体膜组成部分(PGRMCs),是一类小分子蛋白。目前发现有PGMRC1和PGMRC2两种,对PGMRC1的研究较多,它的作用与mPRβ的作用相似,都参与孕酮的调节作用。第3类是孕激素核受体(nPRs),研究发现一些细胞中的孕激素核受体也能够参与介导孕激素的快速生物学反应。本文对介导鱼类非基因作用的孕激素受体在结构、功能和作用机制等方面的研究进展进行了介绍。     

暗纹东方鲀应激逃避行为及其 Mauthner 细胞的形态学观察

探讨暗纹东方鲀(Takifugu obscurus)这种具有胀气、佯死行为的鱼类是否具有快速逃跑行为,以及脑干Mauthner细胞是否是该行为的指令性神经元.研究发现,给予骤发的声音刺激(100 Hz,110 dB),暗纹东方鲀幼鱼和斑马鱼(Danio rerio)均出现快速逃跑行为.但是,与斑马鱼相比,暗纹东方鲀的快速逃跑行为发生概率低,且延迟时间长(P＜0.01).经10 μmol/L GABAA受体抑制剂药浴处理30 min后,与对照组相比,暗纹东方鲀快速逃跑行为发生概率显著增加(P＜0.05),反应延迟时间也显著降低(P＜0.01).组织学观察发现,暗纹东方鲀的Mauthner细胞形态特殊,胞体为椭圆形,细胞的长短轴比显著低于斑马鱼Mauthner细胞的长短轴比,且未见其与第八神经的直接联系.并且,在暗纹东方鲀延脑中未观察到Mid2cm、Mid3cm等神经元.因此,推测由于暗纹东方鲀Mauthner细胞欠发达,导致其快速逃跑能力较弱,故在应激逃避方面,需要进化出其他的防御方式,如胀气等,以保证种群的存活率.本研究为深入研究鱼类的应激反应的生理机制及经济鱼类的健康养殖提供了理论依据.     

共培养系统中4种微藻生态因子的研究

通过二次回归通用旋转组合设计安排实验,研究光强、温度和盐度对分离自对虾池塘水环境中的啮蚀隐藻、新月菱形藻、微绿球藻和蛋白核小球藻的增长率的影响。获得各微藻的最适生态因子及其影响度。啮蚀隐藻的最适生态因子为：光强5750～7944lX,温度21．3～28．3℃,盐度13．3～23．0;影响度依次为：盐度〉温度〉光强。新月菱形藻的最适生态因子为：光强5761～86971X,温度23．4～29．6℃,盐度11．9～25．7;影响度依次为：盐度〉光强〉温度。蛋白核小球藻的最适生态因子为：光强6754～8775iX,温度17．1～20．7℃,盐度19．6～26．4;影响度依次为：温度〉盐度〉光强。微绿球藻的最适生态因子为：光强7128～9012lX,温度18．7～26．7℃,盐度17．9～24．3;影响度为光强〉温度〉盐度。啮蚀隐藻和新月菱形藻的增长率受到以盐度为基础、以光强和温度为协同因子的显著影响。     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Polysaccharolytic activities of bacterial enzymes that degrade the cell walls of Pythium porphyrae, a causative fungus of red rot disease in Porphyra yezoensis

ABSTRACT: Four bacteria with degrading activity against the cell walls of Pythium porphyrae were successfully isolated from Porphyra -culturing environments. The crude enzymes from these bacterial isolates, Bacillus sp. BE1, Bacillus sp. FE1, Pseudomonas sp. PE1, and Pseudomonas sp. PE2, degraded the mycelial cell walls of Pythium sp. N -Acetylglucosamine and glucose were detected in the supernatants of Pythium cell walls treated with the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2 as the final degrading products by high-performance liquid chromatography, whereas only glucose was detected in the supernatant of the cell walls treated with the enzyme from Pseudomonas sp. PE1. Moreover, the activities of β-1,3-glucanase, β-1,3-1,4-glucanase, and chitinase were observed by polysaccharolytic analysis using the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2, whereas only the activities of β-1,3-glucanase and β-1,3-1,4-glucanase were found during analysis using Pseudomonas sp. PE1. β-1,4-Glucanase, β-1,6-glucanase, and mannanase activities were not detected in any of the crude enzymes obtained from these isolates. From the four isolates, the molecular weights (MW) of β-1,3-glucanase and chitinase were estimated to be approximately 50 000–100 000 by the ultrafiltration method. Two Pseudomonas spp. were also suggested to have β-1,3-1,4-glucanases with MW of 50 000–100 000. However, the MW of β-1,3-1,4-glucanases from the two Bacillus spp. might be close to 50 000 or they produce at least two β-1,3-1,4-glucanases with MW of 30 000–50 000 and 50 000–100 000, respectively.     

Development of germ cells and reproductive biology in the sipunculid Phascolosoma esculenta

Sipuncula are of increasing interest for fisheries and aquaculture in China. Sustainable harvests will rely on a better knowledge of reproductive characteristics and stock enhancement. Here, we investigated the structural characteristics of and seasonal changes in germ cell development of the sipunculid Phascolosoma esculenta from the south-eastern coast of Zhejiang, China. An annual survey of egg numbers in the coelom (body cavity) fluid by light and electron microscopy of the females indicates that P. esculenta is dioecious. No defined gonad but dissociated germ cells were found in the coelomic cavity during the 1-year observation. The germ cells showed multiplication and development in the coelomic cavity. Reproduction took place from May to September, with a peak in July and August. The oogenesis can be divided into four phases: cell proliferation, pre-vitellogenesis, vitellogenesis and egg envelope formation and maturation. The process of spermatogenesis can also be divided into four phases: cell multiplication, cell growth, cell maturation and metamorphosis. Monthly changes in the relative number of eggs in each stage indicate that P. esculenta lays eggs in batches. The sperm thrives in the coelomic fluid in the form of cell groups with patterns of genesis and release similar to those of the eggs. Eggs of P. esculenta were fertilized only when reaching the nephridium. The sex ratio was about 1:1 throughout the year.     

虹鳟胚胎受精率适宜发育期的研究

用鉴别液J1、J2与Bouin’s液固定剥膜比较方法,测定了虹鳟(Oncorhynchus mykiss)不同发育期胚胎的受精率。结果表明:用鉴别液J1处理后测得的二细胞期、四细胞期、八细胞期、桑葚期、高囊胚期、原肠中期胚胎的受精率分别为53.21%、77.89%、82.00%、88.2%、87.71%和81.43%;用鉴别液J2处理测得的受精率分别为51.64%、77.86%、81.43%、89.07%、87.50%和81.86%。二者与Bouin’s液固定剥膜后测定的受精率差异不显著(P>0.05),说明这两种鉴别液均可用于生产中测定受精率。在不同的发育时期,鉴别的准确性有所不同,细胞分裂期的准确性远低于桑葚期至囊胚期,这可能是由于胚胎发育不同步和早期胚胎细胞较少所致,因此,鉴别虹鳟鱼卵的受精率最好在桑葚期和囊胚期之间(12～24℃.d)的时间点。     

Establishment,characterization, virus susceptibility and transfection of cell lines from cobia,Rachycentron canadum (L.), brain and fin

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.     

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye,Sander vitreus (Mitchill), at cold temperatures

A cell line, WE‐cfin11e, with an epithelial‐like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast‐like cell line, WE‐cfin11f, and compared with WE‐cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens‐1 (ZO‐1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE‐cfin11e stained for ZO‐1 and only WE‐cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE‐cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE‐cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE‐cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.     

Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata . All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 °C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata .