低等脊椎动物血细胞研究概况

对低等脊椎动物血细胞分类、发生和功能的研究进展进行了综述.低等脊椎动物血细胞分红细胞和白细胞2类.其中白细胞又可分为淋巴细胞、血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞和浆细胞等.有关低等脊椎动物血细胞发生的研究报道较少,并主要集中在鱼类.鱼类血细胞发生的部位包括胸腺、头肾、体肾、脾脏、肝脏和肠粘膜下层等.血细胞的功能与维持动物机体内环境的稳定性有密切关系,低等脊椎动物的血细胞还具有吞噬、分泌等功能,并参与了机体伤口的修复和免疫防御等生理过程.     

长鳍裸颊鲷外周血细胞的显微结构

报道了长鳍裸颊鲷(Lethrinus erythropterus)外周血细胞的显微结构。血涂片经过WRIGHT氏染色,在Olympus BX51显微镜下观察,可鉴别出红细胞、淋巴细胞、嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、血栓细胞和单核细胞等7种类型的血细胞。红细胞数量最多,多为椭圆形,具有圆核;在白细胞中,数量多少依次是血栓细胞、淋巴细胞、嗜中性粒细胞和单核细胞,嗜碱性粒细胞在外周血液中很罕见。血栓细胞体积最小,嗜中性粒细胞最大。淋巴细胞包括小淋巴细胞、中淋巴细胞和大淋巴细胞3种。     

成年似鲇高原鳅外周血细胞及血细胞发生的观察

通过对成年似鲇高原鳅(Triplophysa siluroides)外周血细胞涂片及中肾、肝脏和脾脏组织的印片进行瑞氏(Wright’s)、过碘酸雪夫氏(PAS)和苏丹黑B(SBB)染色,观察各类血细胞形态、大小、分布及细胞化学特征。结果表明:似鲇高原鳅血细胞可分为红细胞、粒细胞、单核细胞、淋巴细胞和血栓细胞,各类血细胞的发生(除血栓细胞)均可分为原始、幼稚和成熟三个阶段。外周血中红细胞比例最多,为97.56%,白细胞中血栓细胞数量最多占52.86%,居白细胞的首位;三种粒细胞中嗜中性粒细胞数量最多占30.25%。粒细胞的主要发生场所在中肾占45.18%,红细胞的主要发生场所在脾脏,占71.62%,肝脏中未见原始血细胞。在中肾中同时可见嗜酸性、嗜碱性和嗜中性三种粒细胞的发生。各阶段的白细胞PAS染色均显示阳性,但只有各阶段的粒细胞SBB染色显示阳性。     

“优鲈1号”大口黑鲈血细胞的形态特征及吞噬功能的研究

用Wright's染色及细胞化学方法,对"优鲈1号"大口黑鲈(Micropterus salmoides)外周血细胞形态特征进行观察,并通过吞噬实验分析其血细胞的免疫功能。结果表明血细胞可分为红细胞和白细胞,白细胞包括粒细胞(Ⅰ型、Ⅱ型和Ⅲ型粒细胞)、单核细胞、淋巴细胞及血栓细胞;其中红细胞数最多,白细胞较少;白细胞中,淋巴细胞比例最大(62.73%)。通过过碘酸-雪夫氏反应(PAS)、苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、过氧化物酶(POX)、酚氧化酶(PO)染色,红细胞染色均呈阴性,而白细胞除AKP、POX染色外,染色均呈阳性,且不同白细胞阳性染色程度不一致,其中血栓细胞染色均较强烈。吞噬实验表明,红细胞具有吞噬功能,吞噬率为(16.17±1.08)%;粒细胞和血栓细胞具黏附能力,且血栓细胞能形成黏附花环,这可能与血栓细胞形态多样性及糖原、脂类、ACP和PO有关。     

军曹鱼稚鱼外周血细胞及其形态学观察

以出膜后第60天的军曹鱼外周血液为材料,利用光镜显微技术研究了其血细胞的种类和形态。外周血液包括5种类型的血细胞,红细胞、淋巴细胞、单核细胞、嗜中性粒细胞和血栓细胞。血细胞计数测得红细胞密度为(2.97±0.82)×109ind.mL-1,白细胞密度为(1.39±0.94)×106ind.mL-1,淋巴细胞、嗜中性粒细胞、单核细胞和血栓细胞在白细胞中所占的比例分别为(53.00±8.51)%、(17.59±4.28)%、(4.78±1.37)%和(24.63±4.08)%。未见到嗜酸性粒细胞和嗜碱性粒细胞。血细胞计数结果表明,红细胞数量远远大于白细胞,白细胞中淋巴细胞数量最多,单核细胞数量最少。实验还观察到处于分裂状态的红细胞、细胞表面有伪足样胞突的小淋巴细胞和细胞表面有许多微绒毛突起的大淋巴细胞、核呈多种形态的单核细胞和中性粒细胞及不同形态的血栓细胞等。     

暗纹东方鲀外周血细胞的显微结构观察

报道了暗纹东方Ｔｕｎ外周血细胞的显微结构。血少片经Ｗｒｉｇｈｔ’ｓ氏染液染色后，可鉴别出红细胞、血栓细胞、淋巴细胞、嗜中性粒细胞、嗜酸性粒细胞。血细胞中，单核细胞数目和粒性细胞数目均很少，血栓细胞数目较多。在肝脏、头肾、脾的涂片中均见到嗜碱性粒细胞。     

厚颌鲂外周血细胞形态及细胞化学特征研究

采用血细胞常规染色和细胞化学染色对厚颌鲂(Megalobrama pellegrini)外周血细胞形态及细胞化学特征进行观察。结果显示:厚颌鲂外周血细胞可分为红细胞、血栓细胞、单核细胞、淋巴细胞和三种类型粒细胞。红细胞有幼稚、成熟和衰老三种形态,其中幼稚和衰老红细胞较少见;血栓细胞数量最多,其次是淋巴细胞。粒细胞中Ⅰ型和Ⅱ型较常见,Ⅲ型粒细胞少有。细胞化学染色显示幼红细胞、血栓细胞和淋巴细胞均呈ACP弱阳性;单核细胞呈ACP和ANAE阳性;Ⅰ型粒细胞是嗜中性粒细胞,POX、ACP和ANAE阳性;Ⅱ型粒细胞是PAS-GL,PAS强阳性和ACP阳性;Ⅲ型粒细胞是Ⅰ型粒细胞的晚期形态。厚颌鲂的PAS-GL可能是嗜酸性/嗜碱性粒细胞,而不是嗜碱性粒细胞的前体。     

黄鳍东方鲀外周血细胞显微结构及血液学指标研究

血涂片经Wright染色液染色,可区分出:红细胞、血栓细胞、淋巴细胞、嗜中性粒细胞、单核细胞等5种血细胞,无嗜碱性粒细胞、嗜酸性粒细胞。白细胞中,血栓细胞数目最多,单核细胞数目最少。与其他硬骨鱼类相比,黄鳍东方鲀的血液性状指标处于较低水平;而雌雄差异不明显。结果可作为黄鳍东方鲀的正常生理常数参考值。     

泥蚶血细胞的形态结构特征及部分免疫功能

利用光镜、透射电镜、扫描电镜及流式细胞仪技术对泥蚶血细胞形态结构以及吞噬活性进行了研究。根据细胞大小、形态结构、颗粒特点,光镜下可将血细胞区分为红色颗粒细胞(占89.67%)、嗜碱性颗粒细胞(占7.05%)、透明细胞(占3.28%)3种主要类型。健康泥蚶血淋巴中血细胞平均密度为(3.29±0.82)×10⁶ /mL。透射电镜下观察到颗粒细胞Ⅰ型、颗粒细胞Ⅱ型、无颗粒细胞;扫描电镜观察下,细胞表面光滑,部分细胞表面有小的突起物。用流式细胞仪检测出P1(13.13%)亚群和P2(78.83%)两个细胞亚群以及没有固定形态的细胞(近8%)。利用APIZYM 试剂盒对健康泥蚶血细胞及血清中的19种酶进行检测,在血细胞中检测到11种酶,在血清中检测到了13种酶,经酵母聚糖刺激后,碱性磷酸酶、酸性磷酸酶、类脂脂酶、N-乙酰葡萄糖胺酶在血细胞和血清中的水平大幅升高。体外吞噬实验观察显示,红色颗粒细胞对酵母聚糖具有明显的吞噬能力,嗜碱性颗粒细胞和透明细胞吞噬作用不显著;在经嗜水气单胞杆菌刺激后,嗜碱性颗粒细胞出现大量聚集现象,透明细胞能吸附颗粒物。吞噬活性研究表明,温度、盐度对泥蚶血细胞的吞噬活性有显著的影响,在泥蚶适宜生长温度(13~30 ℃)区间和最适盐度(20.0~26.2)×10^-12区间,血细胞吞噬活性达到最高值。     

洞庭湖区黄鳝血细胞的分类与计数

采用瑞氏染色和显微镜观察法，对洞庭湖区的黄鳝血细胞进行分类和计数，发现黄鳝的血细胞可分为7类，即红细胞、嗜中性粒细胞、嗜酸性粒细胞、单核细胞、淋巴细胞、血栓细胞和梭形细胞，其中红细胞数目最多，且红细胞数目较其它水生鱼类为高，这可能是与其栖息环境相适应的结果。     

魁蚶各组织溶菌酶活性对鳗弧菌侵染的响应

为了有效防治细菌及其他病原微生物对魁蚶等贝类的危害,本实验研究了魁蚶各组织中溶菌酶活性对鳗弧菌侵染的响应过程,以期探讨魁蚶体内溶菌酶的免疫功能。本实验采用注射活菌的方法侵染20月龄魁蚶个体,随机选取16只个体,在每只个体的斧足处注射1 mL(约1×10~9个)鳗弧菌菌悬液作为感染组;随机选取16只个体不注射鳗弧菌作为对照组。两组魁蚶分别于洁净海水中暂养4、12、24和48 h后,每组随机取4只魁蚶个体的血液、外套膜、鳃、斧足、肝胰腺和闭壳肌等组织,采用ELISA试剂盒测定其溶菌酶含量变化。结果显示,对于鳗弧菌的侵入,魁蚶血液中溶菌酶含量由正常低值迅速增高并一直维持较高的含量,说明血液是魁蚶机体防御病原菌的主要免疫组织之一;魁蚶外套膜在无感染的情况下,对外界水环境的干扰始终保持较高的溶菌酶含量;鳃、斧足的溶菌酶含量均在注射细菌24 h之后明显高于正常值,说明外套膜、鳃、斧足作为魁蚶机体与外界接触的第一道屏障也能应对病原菌入侵,但反应较血液延迟;肝胰腺和闭壳肌的溶菌酶含量变化不明显,推测肝胰腺和闭壳肌不是魁蚶的重要免疫组织或器官。本实验结果可为魁蚶抗病选育及免疫机理方面的研究提供相关的参数。     

Characterization of Glycerophospholipid Molecular Species in Two Species of Arcidaes (Scapharca subcrenata and Scapharca broughtonii)

In this study, the molecular species of glycerophosphocholine (GPCho), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphatidylinositol (GPIns), lysoglycerophosphocholine (LGPCho), and lysoglycerophosphoethanolamine (LGPEtn) from arcidaes Scapharca subcrenata and Scapharca broughtonii were characterized using direct infusion-tandem mass (shotgun) method for the first time. At least 201 and 303 glycerophospholipid (GP) molecular species were characterized, respectively. Most of the predominant GP molecular species contained polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Meanwhile, the arcidae lipids contained a high proportion of polar lipids (81.57–86.72% of total lipids). Among PL, phosphatidylcholine (30.53–32.04 mol%) and phosphatidylethanolamine (21.48–26.71 mol%) were dominant.     

Effects of adrenaline on ionic equilibria in red blood cells of rainbow trout (Salmo gairdneri)

Effects of adrenaline on the equilibrium distributions of Na⁺ , K⁺ , H⁺ , Cl^– , and H₂O across the cell membrane of rainbow trout (Salmo gairdneri) erythrocytes were determinedin vitro, as a function of P CO₂ (1.76–7.77 torr). CO₂-carrying capacity of the blood was also examined. Plasma catecholamine concentrations inunanaesthetized, unrestrained trout were 3.1 nM adrenaline and 1.2 nM noradrenaline. Elevation of the plasma adrenaline concentrationin vitro to 4.6 × 10³ nM resulted in net gains of Na⁺ , Cl^– and H₂O by red cells, a net loss of H⁺ from red cells, and a pronounced red cell swelling. Adrenaline also reduced the CO₂-carrying capacity of trout bloodin vitro. The magnitudes of these effects increased with P_CO2 and, thus, were sensitive to blood HCO₃ ^– concentrations. The distribution of K⁺ between red cells and plasma was unaffected by adrenaline. Adrenergic-mediated ion movements and red cell swelling were sensitive to both propranolol and SITS. These results are consistent with the symport NaCl uptake model for adrenergic-mediated swelling of Baroinet al. (1984). The adrenergic response of fish erythrocytes may function to ameliorate the effects of blood acidoses on O₂-carrying capacity by maintaining red cell pH in the face of a decrease in plasma pH.     

魁蚶过氧化氢酶基因克隆及表达分析

采用RT-PCR和c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)克隆出魁蚶过氧化氢酶(Sb CAT)基因c DNA全长序列,该基因全长为2181 bp,包括1431 bp的开放阅读框(open reading frame,ORF),96 bp的5'端非翻译区(UTR)和654 bp的3'-UTR。其中ORF编码477个氨基酸,预测分子量为54 ku,理论等电点为8.03。Sb CAT氨基酸序列与其他所选动物的氨基酸序列具有较高的相似性,其相似度为68%~96%,Sb CAT氨基酸具有CAT基因家族的特征性序列,包括CAT活性位点,1个亚铁血红素结合位点及3个催化位点残基。此外,Sb CAT还具有保守的亚铁血红素结合口袋与还原型辅酶Ⅱ(NADPH)结合位点。采用实时荧光定量PCR(q RT-PCR)检测了Sb CAT的组织表达特征。结果显示,Sb CAT m RNA在所检测的6种组织中均有表达,在外套膜中表达量较高,在肝胰腺和红细胞中表达量较低。经鳗弧菌和金黄色葡萄球菌刺激后,鳗弧菌刺激组的外套膜表达量一直较低,其他组织均表现出先上升再下降的趋势。研究表明,Sb CAT可能在魁蚶免疫防御中发挥重要作用。     

辽宁沿海5个毛蚶群体遗传多样性的ISSR分析

采用ISSR技术,对辽宁沿海5个毛蚶群体的遗传多样性进行了研究。在选择的12个随机引物中共检测到137个扩增片段,长度在200~2 500 bp,每个个体获得的扩增条带在9~15条不等。利用POPGEN 3.2和MEGA 2统计软件,获得实验数据,确立了5个群体间的亲缘关系。结果表明,毛蚶5个地理群体分化不明显,没有形成不同的地理种群;聚类分析显示,丹东和庄河亲缘关系最近,然后依次为金州、营口、锦州;5个群体无论是在多态位点比例还是在平均杂合度上都处于较高水平,说明毛蚶当前种质资源状况良好,遗传多样性水平较高。     

锦鲤墨蝶呤还原酶基因的克隆、表达和定位分析

为探究SPR在锦鲤体色形成中的作用,实验利用RACE技术获得spr cDNA全长序列,并分析其时空表达模式,同时利用Western blot和免疫组织化学方法检测SPR蛋白在皮肤、鳍条和鳞片中的分布和表达情况。结果显示,spr cDNA全长879 bp,包含132 bp和134 bp的5′和3′非编码区,开放阅读框510 bp,编码170个氨基酸残基。氨基酸序列比对和系统进化树分析显示,锦鲤SPR具有保守的adh_short_C2结构域,与金鱼相似性高达97.7%。spr在各组织中均有表达,其中皮肤的表达量最高。spr在锦鲤个体发育的4个阶段表现为先降后升。纯红、纯白及红白3种体色锦鲤皮肤、鳞片和鳍条中spr mRNA和蛋白表达水平基本一致,在纯红锦鲤皮肤中表达量最高,红白锦鲤白色皮肤、鳞片和鳍条的表达量最低。SPR组织定位分析显示,红色锦鲤和白色锦鲤皮肤中均检测到阳性信号,其中红色皮肤阳性信号强度高于白色皮肤。研究表明,spr可能与锦鲤红/黄色素细胞的分化和形成具有一定相关性,参与了锦鲤体色的形成。     

文蛤浮游期发病幼虫细菌群落分析和致病菌的分离鉴定

为研究文蛤育苗过程中幼虫病害及其主要致病菌，实验通过构建细菌16S rDNA克隆文库、病原菌分离纯化、人工感染和药敏实验等方法对育苗场发病的浮游期文蛤壳顶幼虫样品进行了系统分析。结果显示，发病的文蛤壳顶幼虫细菌群落多样性低，地中海弧菌占比高达75%以上，推断其可能为引发此次幼虫发病的主要致病菌。从发病幼虫的匀浆组织中分离获得该优势菌株，测序及系统发育鉴定为地中海弧菌。人工感染实验确定了其致病性，菌株US2-01在1.0×10⁶ CFU/mL的菌液浓度下浸泡感染文蛤浮游幼虫，96 h累计死亡率为84%。药敏实验表明，地中海弧菌菌株US2-01在12种抗生素的测试中对常用的青霉素、氨苄西林、红霉素等抗生素具有一定的耐药性，对四环素和多西环素中度敏感，对头孢他啶、庆大霉素、卡那霉素等其余7种抗生素呈现高度敏感。本研究首次报道了地中海弧菌为文蛤浮游期幼虫致病的一种潜在病原，研究结果可为文蛤幼虫疾病研究及贝类苗种培育过程中的病害防控提供科学依据。     

Hematological Parameters and Blood Cell Morphology of Male and Female Schizothorax (Racoma) davidi (Sauvage)

The aim of this study was to obtain a basic knowledge of the hematology of Schizothorax (Racoma) davidi (Sauvage). The results showed that red blood cell and white blood cell counts as well as mean cellular hemoglobin concentration were significantly higher in females than in males, while mean cell volumes were significantly lower in females than in males. No significant differences were seen in other hematological parameters in relation with gender. Under light microscope, erythrocytes (mature and immature), leukocytes (neutrophils, lymphocytes, and monocytes), and thrombocytes were distinguished. Mature erythrocytes were ellipsoidal cells with oval, central nucleus. Neutrophils contained banded or segmented nuclei. Small lymphocytes had a very thin rim of basophilic cytoplasm. Monocytes often possessed vacuoles in their cytoplasm. Thrombocytes existed in several forms with relatively large nucleus surrounded by a pink‐stained or hyaline cytoplasm. The percentage of the different leukocytes revealed predominance of neutrophils and small lymphocytes, followed by monocytes and large lymphocytes. The neutrophil percentages of each stage showed that metamyelocyte accounted for the most, followed by banded neutrophils. This investigation could be used for evaluation of the health status of cultured male and female S. davidi and will provide early detection of clinical pathology.     

Preclinical toxicity and safety evaluation of Althaea officinalis L. extract as naturopathic medicine in common carp (Cyprinus carpio L.): Hematological and biochemical study

ABSTRACT

This study was conducted to evaluate the effects of marshmallow extract (Althaea officinalis L.) administration on blood cells and biochemical parameters of carp liver. A total of 150 carps (Cyprinus carpio, initial body mass of 37.7 ± 4.4 g) were fed diets containing 0.0 (control diet), 2.5, 5, and 10 g marshmallow extract for 60 days. On days 30 and 60 of the experiment blood samples were collected and hematological parameters and liver enzyme activities—aspartate aminotransferase (AST), alanine amino transferase (ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP)—were measured. No significant difference was found (p > .05) in red blood cells (RBC); hemoglobin concentration (Hb); hematocrit (Htc); mean corpuscular volume (MCV); mean corpuscular hemoglobin concentration (MCH); or the percentage of neutrophils, basophils, and eosinophils in fish fed with different concentrations of marshmallow extract on days 30 and 60. However, the white blood cell (WBC) count was increased significantly in the fish fed for 60 days with feed supplemented with 5 g marshmallow extract (p < .05). The MCHC was increased signi?cantly in ?sh fed with diets supplemented with 2.5 g marshmallow extract for 60 days (p < .05). Administration of marshmallow extract (5 g) significantly increased (p < 0.05) lymphocytes on days 30 and 60. However, dietary intake of 10 g marshmallow extract had significantly decreased the monocyte percentage after 60 days. A significant increase was observed in AST, ALT, ALP, and LDH levels in livers of fish fed with extract 10 g, which may be attributed to cytotoxicity. However, marshmallow extract 2.5 and 5 g did not have adverse effects on liver enzymes. These results indicate that preclinical dietary supplementations of 2.5 and 5 g marshmallow extract per kg diet have no side effects on common carp.