斑马鱼卵子中母源性补体成分的检测及溶菌活性研究

本研究首次检测到斑马鱼(Danio rerio)受精卵中存在母源性补体成分C3和Bf。在斑马鱼卵子胞浆中加入适量C3(所有补体激活途径的关键因子)抗体使补体C3沉淀失活,或将卵子胞浆45℃水浴30min(热灭活补体),均可显著降低卵子胞浆的抑菌率,说明斑马鱼卵子胞浆中的补体系统具有免疫活性。在此基础上,通过补体激活途径的抑制实验研究了母源性补体的作用方式。向斑马鱼卵子胞浆中分别加入适量C1q(经典途径关键成分)抗体和C4(同时参与经典途径和凝集素途径)抗体对其抑菌率无显著影响,而加入Bf(替代途径关键成分)抗体却能够显著降低卵子胞浆的抑菌率。另外,在斑马鱼卵子胞浆中加入EGTA螯合Ca2+抑制经典途径和凝集素途径,卵子胞浆的抑菌率并无明显变化,而加入EDTA同时螯合Ca2+和Mg2+使替代途径也受到抑制后,溶菌率则显著降低。补体途径抑制实验证明,斑马鱼早期胚胎中的补体成分主要是通过替代途径发挥作用。     

棘皮动物补体系统的研究进展

研究棘皮动物的补体系统对于探索棘皮动物的防病抗病机制及研制免疫药物具有极其重要的意义。文章从棘皮动物类补体物质的种类、功能及作用机制等方面总结了国内外近几年来对棘皮动物补体系统的研究进展,指出了研究棘皮动物补体系统的现实意义。     

海洋无脊椎动物浮游幼虫的多样性

<正>海洋是一个巨大的生命空间。根据R.D.Barnes(1968)的动物分类系统(修改),动物界计有37个门,其中34个门为无脊椎动物,31个门为海洋无脊椎动物。据估算,全球海洋无脊椎动物约有14万多种。31个门海洋无脊椎动物中,原生动物为单细胞动物。本文仅就30门生活在海洋中的多细胞无脊椎动物其胚后发育中具有浮游幼虫阶段的门类的幼虫及其早期发育作简要论     

饲用枯草芽孢杆菌HGcc-1对鲤肠肝健康、血清补体及肠道菌群的影响

为了研究枯草芽孢杆菌HGcc-1对鲤肠肝健康、血清补体和肠道菌群的影响,实验选择体质量为(13.10±0.39) g健康鲤,随机分为HGcc-1添加组和对照组,养殖20周后测定生长指标,用试剂盒检测了鲤血清内毒素(LPS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、总补体和溶菌酶,并对肠道菌群进行了16S rRNA测序,同时将添加组和对照组的肠道菌群转接至无菌斑马鱼,然后检测无菌斑马鱼体内毒素结合蛋白(LBP)、ALT、AST的水平和C3、C4基因表达量;最后还检测了HGcc-1直接作用于无菌斑马鱼时,无菌斑马鱼的ALT、AST的水平和C3、C4基因表达量。结果显示,HGcc-1对鲤的增重率无显著影响;与对照组相比,饲料中添加HGcc-1显著降低鲤血清内毒素、ALT和AST水平;同时HGcc-1添加组血清总补体水平显著升高。在门水平上,HGcc-1添加组中梭杆菌门丰度比对照组增加了47.1%;变形菌门丰度比对照组减少了70.7%;在属水平上,HGcc-1添加组中鲸杆菌属(的丰度比对照组增加了47.1%;柠檬酸杆菌属和气单胞菌属(的丰度比对照组分别减少了56.6%和70.9%。利用无菌斑马鱼模型进一步发现,HGcc-1添加组鲤肠道菌群显著降低了无菌斑马鱼LBP含量和AST水平,显著上调了无菌斑马鱼补体C3和C4基因表达;与此同时,HGcc-1与无菌斑马鱼直接互作也降低了鱼体ALT和AST水平。研究表明,饲料中添加枯草芽孢杆菌HGcc-1能够改善鲤肠肝健康、血清补体以及肠道菌群稳态。本研究为枯草芽孢杆菌HGcc-1的下一步应用奠定了理论基础。     

酵母多糖、壳聚糖、甘草酸对草鱼非特异性免疫功能的影响

将草鱼放入网箱中,在基础饲料中分别添加不同剂量的酵母多糖、壳聚糖和甘草酸后,进行投喂,研究其对草鱼血清中菌酶(LSZ)、过氧化氢酶(CAT)及补体C3活性的影响。结果表明:投喂添加中药免疫添加剂的饲料49d后,与对照组比较,酵母多糖添加剂各组、壳聚糖高剂量组草鱼血清LSZ活性显著提高(P0.05);酵母多糖低剂量组、中剂量组和壳聚糖高剂量组草鱼血清CAT含量显著提高(P0.05);酵母多糖中剂量组草鱼血清补体C3活性显著提高(P0.05),说明中药免疫添加剂对增强草鱼免疫活性具有一定的作用。     

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。     

鱼类补体系统（Complement system of fish）的特点

本刊讯：鱼类补体系统由35种以上蛋白质裂解酶、酶抑制因子和受体构成。浙江大学动物科学学院张静等人研究报道，与哺乳动物相比，硬骨鱼类补体系统具有以下特点：     

三种鲟鱼血清及组织中磷酸酶和补体活性的比较

对施氏鲟、小体鲟和西伯利亚鲟的血清、肝脏、鳃及肠中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、补体C3及补体C4活性进行了测定和比较.AKP活性在小体鲟鳃和肠中显著高于施氏鲟和西比伯利亚鲟(P＜0.05),而其在三种鲟鱼的肝脏和血清中差异不显著(P＞0.05);ACP活性在小体鲟肠中显著高于施氏鲟和西比伯利亚鲟(P＜0.05),而其在三种鲟鱼的肝脏、鳃和血清中差异不显著(P＞0.05);补体C3活性在小体鲟肝脏和肠中显著高于施氏鲟和西比伯利亚鲟(P＜0.05),而其在三种鲟鱼的鳃和血清中差异不显著(P＞0.05);小体鲟血清中补体C4活性显著高于施氏鲟和西伯利亚鲟(P＜0.05),而补体C4活性在三种鲟鱼的肝脏、鳃及肠中差异不显著(P＞0.05).由此可见,虽然三种同为鲟科鲟属鱼类,但它们之间的AKP、ACP、补体C3及补体C4活性存在着种间差异.     

施氏鲟细菌感染应答中几种血清免疫因子的变化

本文研究嗜水气单胞菌(Aeromonas hydrophila,Ah)感染对1+龄施氏鲟(Acipenser schrencki)血清中补体、一氧化氮(NO)水平及一氧化氮合酶(NOS)活性的影响,以探讨鲟科鱼类细菌感染应答的敏感因子。首先采用腹腔注射法测定了Ah对施氏鲟的LD50,然后给健康非免疫状态的1+龄施氏鲟,注射LD50剂量Ah菌液,观察发病情况,同时采集健康组和病理模型组血清及肝组织样品,测定血清中补体、NO水平及NOS活性。结果显示:施氏鲟在感染Ah时,血清中补体C3、C4的含量均有一定程度升高,其中C3的含量显著升高,表明C3在施氏鲟对抗细菌感染中起重要作用。NO是感染应答中重要的炎性反应调节分子,在Ah感染后,施氏鲟血清中NO的水平显著升高,肝脏中的NO水平变化不显著。NOS在NO的生成中起关键作用,但施氏鲟感染后血清及肝脏中的NOS活性无显著变化,由于未做分型检测,属正常现象。     

茯苓多糖对中华鳖非特异性免疫功能的免疫调节作用

研究了口服茯苓多糖对中华鳖(Trionyxsinensis)非特异性免疫功能的影响。试验共分3个组,在基础饲料中添加20·0mg/kg茯苓多糖作为试验组,添加500·0mg/kg酵母细胞壁作为免疫刺激剂对照组,以投喂基础饲料作为空白对照组。连续投喂28d后,测定中华鳖血液中白细胞的吞噬活性、血清溶菌活力和补体活性。结果表明:在中华鳖饲料中添加茯苓多糖和酵母细胞壁能显著提高中华鳖白细胞的吞噬活性(t检验,P<0·05),而2个试验组之间中华鳖血液中白细胞的吞噬活性没有显著差异(t检验,P>0·05);2个试验组中华鳖血清中补体C3和C4的活性与对照组之间呈显著差异(t检验,P<0·05),而试验组之间无显著性差异(t检验,P>0·05)。本研究结果说明在中华鳖饵料中添加茯苓多糖和酵母细胞壁对中华鳖的非特异性免疫功能有增强作用。     

High stocking density produces crowding stress altering some physiological and biochemical parameters in gilthead seabream, Sparus aurata, juveniles

High stocking density has been shown to produce a wide variety of effects on cultured fish populations, such as alterations in behavior and poor feed utilization, resulting in mortality and poor growth. High stocking density has also been reported to produce chronic stress and mobilization of energy sources in fish. There are few studies focusing on the effect of high stocking density on one of the most important marine fish species for Mediterranean aquaculture, gilthead seabream (Sparus aurata). Consequently, this study investigates the effect of high stocking density on juveniles of this species, focusing on effects of stocking density on growth, biochemical composition, immune status and hematology.The results of this study showed that high stocking density produced a chronic stress situation. Plasma cortisol levels increased up to 16.25 ng ml–1 in fish held at high stocking; this value was 4 fold that of fish held at low stocking; density (3.91 ng ml–1 ). As a consequenceof this stressful situation, there was both haemoconcentration and a decrease in alternative complement pathway (ACP), an important component of the immune system of fish (from 167.23 U ml–1 down to 146.37 U ml–1). Haematocrit, haemoglobin concentration and red blood cell count were significantly higher in fish held at high stocking density (43.87%, 10.76 9dl–1 and 3.36 ×106 mm–3, respectively) compared with those fish held at low stocking density (37.21%, 9.32 g/dlg–1 and 2.82 ×106 mm–3, respectively).In addition, high stocking density produced a decrease in hepatosomatic index (from 2.26 down to 2.04) and altered liver fatty acid composition. Oleic acid (18: 1n-9) decreased in liver total lipids of fish held at high stocking density and arachidonic acid (20: 4-n6) and n-3 high unsaturated fatty acids (n-3 HUFA) were reduced in liver polar lipids of those fish. These alterations reflect the effect of stocking density on lipid metabolism to help meet the increased energy demand.     

仿刺参补体B因子溶血活性初步研究

本文应用高等哺乳动物血清补体B因子溶血检测技术检测仿刺参个体体内补体B因子。通过溶血法检测了健康仿刺参以及人工诱导化皮仿刺参体腔液补体B因子活性。结果显示仿刺参体腔液有补体B因子存在，且含补体B因子仿刺参体腔液溶血活性明显高于去补体B因子体腔液的溶血活性。实验对仿刺参体腔液进行加热处理结果显示其补体B因子是对热不稳定的分子，此特性与高等哺乳动物B因子特征相同。仿刺参体内存在补体B因子，它与脊椎动物补体B因子性质相似，在免疫旁路途径中起重要作用。     

Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L.

Nucleotides from yeast RNA were evaluated for their ability to enhance the non-specific immune responses in carp, Cyprinus carpio . Oral administration of nucleotides to fish daily for 3 days resulted in enhanced responses of phagocytic and nitroblue tetrazolium (NBT) activities in kidney phagocytic cells. This activation of kidney cells was observed for at least 10 days post-treatment. The serum complement and lysozyme activities also increased in fish treated with nucleotides. Furthermore, the number of Aeromonas hydrophila in nucleotide-treated fish significantly decreased in the blood, kidney and liver after intraperitoneal injection. Thus yeast nucleotides appear to enhance non-specific immune responses in fish.     

补体和溶菌酶在清除文昌鱼体内细菌中的作用

文昌鱼作为研究脊椎动物起源和进化的模式生物,由于缺乏自由循环的血细胞,关于其清除体内病原体的机制,迄今尚无报道。采用体内细菌清除试验、体外杀菌试验、特异性抗体抑制试验、抑制补体途径试验及Western blot方法,研究了文昌鱼体内细菌清除机制及动力学。试验结果表明,文昌鱼能有效清除入侵的异源微生物,体液对大肠杆菌具有较强的杀灭活性。补体和溶菌酶是参与清除入侵大肠杆菌的主要因子,通过替代途径激活的补体较溶菌酶作用更强,这是关于文昌鱼清除体内细菌机制及其相关因子的首次报道。     

海参防御机制的研究进展

海参乃棘皮动物门，海参纲动物的统称，全世界海参约有1200种。在我国海域分布的有140多种，其中品质最佳、药用价值最广的是产于中国辽宁、山东、河北等北太平洋沿海的仿刺参（Apostwhopus japonicus）。研究表明，在海参体壁中含有丰富的胶原成分和蛋白聚糖，并含有钙、镁盐及铁、锰等多种微量元素。     

Enzyme treatment of Trypanosoma danilewskyi (Laveran and Mesnil) increases its susceptibility to lysis by the alternative complement pathway of goldfish, Carassius auratus (L.)

This study examined whether in vitro-cultured Trypanosoma danilewskyi were susceptible to lysis in the presence or absence of anti-parasite antibodies and complement. Cultured trypanosomes were resistant to lysis by either immune or non-immune goldfish serum. However, trypanosomes treated with the proteolytic enzyme trypsin, which destroys surface proteins of the parasites, became susceptible to lysis when exposed to either immune or non-immune goldfish serum. The lysis by goldfish serum was dependent on the presence of heat-labile factors and occurred at 4 and 20 degrees C. The lysis was also dependent on the presence of Mg(2+) ions but not Ca(2+) ions. Furthermore, treatment of the parasites with different sialidases did not enhance their susceptibility to lysis by goldfish serum. Trypsinized parasites regained resistance to lysis after at least 6-h cultivation in the absence of trypsin and the restoration of full resistance was observed after 24-h cultivation. The resistance to lysis was abrogated when the protein synthesis inhibitor, puromycin, was added to the cultures. These results suggest that trypsinized trypanosomes were susceptible to lysis by goldfish complement (alternative pathway) and that protective surface proteins of the parasite were required for the resistance of normal trypanosomes to lysis.     

盐酸小檗碱对草鱼补体系统及补体c3作用的研究

为了探究盐酸小檗碱(berberine hydrochloride)对草鱼(Ctenopharyngodon idellus)补体c3的作用机理,用含0、0.06%、0.12%、0.24%盐酸小檗碱饲料投喂草鱼,在第7 d、14 d检测血清中补体c3含量,在第14 d检测肝脏中补体c3 mRNA表达量及急性感染嗜水气单胞菌(Aeromonas hydrophila)后存活情况。并进一步对盐酸小檗碱体内药代动力学和补体消耗试验进行了研究。结果显示;摄食盐酸小檗碱的草鱼血清中补体c3含量和肝脏补体c3 mRNA表达量均显著性增加(P0.05/P0.05),草鱼的存活率与对照组相比也有显著性提高(P0.01)。补体消耗试验结果显示,当盐酸小檗碱在血清中浓度小于5 mg/L时与对照组相比补体消耗不显著,当盐酸小檗碱浓度大于5 mg/L时,补体消耗达到显著性差异(P0.05),表明此时盐酸小檗碱与补体c3直接结合,激活补体。药代动力学实验发现,灌服盐酸小檗碱后,出现双峰现象,两峰值分别是0.243 mg/L和0.117 mg/L,此浓度远远低于与补体c3直接结合量最低量(5 mg/L)。结果表明,盐酸小檗碱对草鱼补体c3的作用机理可能是通过上调补体c3mRNA的表达增加补体c3数量,而非通过直接结合补体c3来改变其活性。     

Investigation of humoral immune factors from selected groups of southern bluefin tuna, Thunnus maccoyii (Castelnau): implications for aquaculture

Serum immunoglobulin, lysozyme and classical and alternative complement activity were analysed in different groups of wild and captive southern bluefin tuna (SBT), Thunnus maccoyii (Castelnau), from ambient water temperatures of 12 ± 1 and 20 ± 1 °C. Groups held captive for the longest time were found to have the highest levels of these humoral immune mediators, despite a drop in ambient temperature from 20 ± 1 to 12 ± 1 °C during the captivity period. Therefore, it may be that the immune response in these endothermic fish is not inhibited by low temperature to the extent seen in poikilothermic fish. Also, length of time in captivity appears to be associated with increased antigen exposure to maintain high levels of humoral immune mediators in these groups. Lysozyme activity was optimal at pH 5.8 and 6.2, suggesting that two isoforms, with different pH optima, are present. The SBT serum was found to lyse sheep erythrocytes by both classical and alternative complement pathways. Classical pathway activity occurred in the absence of prior sensitization with antiserum to sheep red blood cells, suggesting that natural antibodies may be present (or lectin or C-reactive protein mediated activation). Complement activity was relatively resistant to freezing at −20 °C but heating at between 45 and 50 °C for 20 min destroyed all complement activity.