流式细胞术在克氏原螯虾血细胞的分类、活性和免疫功能研究中的应用

应用流式细胞术(FCM)对克氏原螯虾(Procambarus clarkii)血细胞的分类、活性和免疫功能进行了研究。结果显示:血细胞可分为透明细胞、小颗粒细胞和大颗粒细胞三个亚群,组成比例分别为(26.25±5.29)%、(51.44±7.02)%和(11.20±1.82)%;螯虾血细胞的平均总凋亡率约为3.12%;血细胞对荧光大肠杆菌的吞噬活力显著(P<0.05)高于荧光微球,吞噬率分别为17.04%和14.57%;血细胞在自然生理状态下含有一定量的活性氧,其在两类颗粒细胞的含量显著(P<0.05)高于透明细胞,在大颗粒细胞中最高。结果表明,FCM能较好地应用于虾类的血细胞分类和功能研究。     

黑褐新糠虾血细胞形态及其与日本新糠虾对溶藻弧菌敏感性的比较

实验采用Giemsa与Wright' s两种染色方法对黑褐新糠虾血细胞进行染色,根据血细胞大小,所含颗粒与否,颗粒多少和核质比将其分型,并对各型血细胞所占比例进行统计.结果表明,通过比较Giemsa染色与Wright's染色两种方法的染色时间和效果,Wright's染色更加适合糠虾血细胞.通过染色,将黑褐新糠虾血细胞分为3种类型,它们分别为透明细胞、半颗粒细胞和颗粒细胞.它们的胞体大小依次增大,核质比依次降低.对此3种类型血细胞比例分别统计后发现,颗粒细胞所占比例最多约( 48.38％±0.05％),其次是半颗粒细胞约为(35.22％±0.04％),最少的为透明细胞约为(16.40％±0.01％).对不同浓度溶藻弧菌的感染试验发现,日本新糠虾在较短的时间内出现死亡,即48h,而黑褐新糠虾72 h出现死亡,各浓度组中日本新糠虾死亡率均高于黑褐新糠虾.黑褐新糠虾相对日本新糠虾来说,对溶藻弧菌具有较低的敏感性,可能与其血细胞中颗粒和半颗粒比例较高有关.     

绢丝丽蚌血细胞类型的研究

依据是否有伪足、细胞质中有无颗粒和细胞大小对绢丝丽蚌血细胞进行了分类.结果表明:绢丝丽蚌血细胞分为无颗粒细胞、颗粒细胞、透明细胞和类淋巴细胞4种类型,各有不同结构,并协同行使机体的运输、防御和免疫功能.     

橄榄蛏蚌性腺发育与生长

2008年8月—2009年10月,每月采集一批橄榄蛏蚌,分别进行可量性状的测定和年龄鉴定,并研究了其肥满度周年变化规律。为了揭示其繁殖特性,取繁殖季节性腺制作石蜡切片,经苏木精和伊红染色后在显微镜下观察其形态结构,并测量性腺细胞的直径。结果表明,橄榄蛏蚌的肥满度在每年的5月出现最高峰,且一年只有一个高峰,因而橄榄蛏蚌繁殖为一年产卵一次、繁殖一次。橄榄蛏蚌性腺细胞直径为49.3～70.2μm。随着水温的升高,橄榄蛏蚌的摄食活性增强。统计学数据分析表明,橄榄蛏蚌体质量与壳长、壳宽和壳高均呈指数关系,而后三者之间呈线性关系。对体质量影响最大的指标是壳高,最小的指标为壳长。     

流式细胞术比较研究三种对虾血细胞的分群

应用流式细胞术对墨吉对虾(Penaeus merguiensis)、南美白对虾(Penaeus vannamei)、斑节对虾(Penaeus monodon)三种对虾的血细胞类型进行了比较研究。根据其血细胞的前向角散射光(FSC)和侧向角散射光(SSC)特征的不同将血细胞分群,FSC和SSC二维图分析表明三种对虾的血细胞都可分为三个亚群:透明细胞、小颗粒细胞、大颗粒细胞。同时还对各种类型血细胞所占比例进行了比较研究,发现各个亚群血细胞所占组成比例在三种对虾之间具有相似性。其比例均为:小颗粒细胞最多,约为45%~49%;透明细胞次之,约为26%~28%;大颗粒细胞最少,约为10%~12%。     

凡纳滨对虾不同类型血细胞的活性氧水平和酯酶活性

应用流式细胞术(FCM)测定凡纳滨对虾(Litopenaeus vannamei)不同类型血细胞(透明细胞、小颗粒细胞和大颗粒细胞)的非诱导性和诱导性活性氧(ROS)含量以及非特异性酯酶活性,探讨不同类型血细胞的免疫功能.结果显示,透明细胞的非诱导性ROS含量最低,显著低于小颗粒细胞和大颗粒细胞(P＜0.05),大颗粒细胞的非诱导性ROS含量最高;经脂多糖(LPS)刺激后,透明细胞的ROS含量没有显著变化(P＞0.05),小颗粒细胞和大颗粒细胞的ROS含量显著提高(P＜0.05);大颗粒细胞的酯酶活性显著高于小颗粒细胞和透明细胞(P＜0.05).研究表明,3类血细胞在活性氧含量和酯酶活性的功能上存在差异,两类颗粒细胞的活性氧水平较高,大颗粒细胞的酯酶活性较强.     

温度对橄榄蛏蚌滤水率的影响

在实验室条件下,测定不同温度下橄榄蛏蚌(Solenaiaoleivora)的滤水率。结果表明,在15℃、20℃、25℃、30℃4个温度梯度下,橄榄蛏蚌的滤水率呈一个峰值变化,滤水率最大值出现在20℃,最小值出现在30℃;对于大规格和小规格橄榄蛏蚌,最大值分别为0 2388L/(ind·h)和0 1353L/(ind·h),最小值分别为0 0607L/(ind·h)和0 0358L/(ind·h)。方差分析(ANOVA)表明,温度和个体大小对橄榄蛏蚌的滤水率有极显著影响(F>F0 01),并且不同规格的橄榄蛏蚌,小规格组滤水率大于大规格组滤水率。此结论与多数双壳贝类的生理现象一致,即在一定温度范围内,温度越高,生理活动越旺盛;温度过高或过低均能使生物生理活动能力降低。     

白洋淀日本沼虾血细胞的初步研究

对取自华北白洋淀种群的日本沼虾(Macrobrachium nipponense)血细胞进行显微形态观察、分类、测量和计数。依据细胞的形态,胞质中颗粒的数量和密度以及细胞的核质比把日本沼虾血细胞分为透明细胞(hyaline cell,HC)、半颗粒细胞(semigranular cell,SGC)和颗粒细胞(granular cell,GC)3类。血细胞大小依次为颗粒细胞>半颗粒细胞>透明细胞。血细胞密度为(1.13±0.50)×106个/mL,其中GC占48.5%,SGC占31.8%,HC占19.7%。并对我国日本沼虾的华南种群、华中种群及白洋淀所在地华北种群的血细胞大小和数量进行了比较。     

成年日本新糠虾血细胞组成、形态及其不同发育阶段的免疫活性酶

采用光镜和电镜的方法,以血细胞所含颗粒与否,颗粒多少和核质比等将成年日本新糠虾血细胞进行分型,并对各型血细胞所占比例进行统计。利用生化分析的方法,对不同发育阶段(新孵后0、10、20和30d)日本新糠虾全组织中磷酸酶(酸性磷酸酶和碱性磷酸酶)、溶菌酶和酚氧化酶的活性进行测定。结果表明,日本新糠虾血细胞可为3种类型,它们分别为透明细胞、半颗粒细胞和颗粒细胞。它们的胞体大小依次增大,核质比依次降低。对此3种类型血细胞比例分别统计后发现,颗粒细胞所占比例最多约为47.53%±0.02,其次是半颗粒细胞约为31.23%±0.01,最少的是透明细胞约为21.24%±0.02。日本新糠虾体内磷酸酶(酸性磷酸酶和碱性磷酸酶)、溶菌酶和酚氧化酶的活性均有随个体发育而呈降低的趋势。3种酶中以溶菌酶的活性最高,平均约为305.20U/mg蛋白,其次是酸性磷酸酶约为0.2365U/mg蛋白,而酚氧化酶和酸性磷酸酶在成体中的活性基本一致,约为0.1295U/mg蛋白。研究表明,日本新糠虾血细胞组成中,以颗粒细胞为主。溶菌酶是其主要的免疫活性物质。日本新糠虾体内磷酸酶(酸性磷酸酶和碱性磷酸酶)、溶菌酶和酚氧化酶活性有随其个体发育而逐渐...     

克氏原螯虾血细胞及免疫功能的初步研究

甲壳动物血细胞在宿主免疫应答中发挥着重要的作用，其中包括识别、吞噬、包囊黑色素形成、胞毒作用和胞间的信息传递。不同类型的血细胞其防御机制不同，因此血细胞的形态和分类是甲壳动物免疫学的基础。通常情况，十足目甲壳动物的血细胞类型主要依据细胞质中颗粒物质的有无和颗粒物质的相对大小进行分类。研究采用Giemsa染色法、H.E染色法和电子显微技术法对克氏原螯虾血细胞进行分类，观察到3类血细胞：透明细胞、小颗粒细胞、大颗粒细胞，它们所占的比例分别为19.9%，46.1%，34.1%。在电镜下，这3类血细胞的超微结构极为容易辨识。透明细胞通常在3类细胞中最小，核质（N/C）比最高，但无明显的颗粒；相比之下，小颗粒细胞和大颗粒细胞内部的颗粒数量逐渐增多。利用体内和体外培养技术研究克氏原螯虾血细胞对外源体（以Sephadex A-25色谱硅珠作为抗原）的包囊作用，初步探讨克氏原螯虾血细胞在对外源体免疫应答过程中的识别机理。将Sephadex A-25色谱硅珠植入克氏原螯虾体内，抗原物周围被大量血细胞包囊，通过切片技术观察到抗原物周围的血细胞绝大部分为小颗粒细胞，证实在包囊过程中起主要作用的血细胞是小颗粒细胞，即小颗粒细胞在包囊反应中比较活跃。研究结果为以后进一步研究克氏原螯虾的细胞免疫机制和提高其抗病能力提供了科学依据。     

In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes

Bivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNI production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters.     

Flow cytometric studies on the morphology and immunological functions of hemocytes in the Iwagaki oyster Crassostrea nippona

The Iwagaki oyster Crassostrea nippona may represent an aquaculture alternative to the Pacific oyster in Korea, Japan and China. In this study, morphology and immune-related functions of hemocytes of C. nippona were characterized using light microscopy and flow cytometry. Both light microscopy and flow cytometry indicated that C. nippona hemocytes are composed of three different types: granulocytes, hyalinocytes, and blast-like cells. Hyalinocytes were the most abundant and intermediate-sized cells, while granulocytes were the largest cells, containing many granules in the cytoplasm. Blast-like cells were characterized by a smaller size and very thin cytoplasm. Granulocytes were mainly engaged in phagocytosis and oxidative activities, although hyalinocytes also exhibited a certain level of immune-related activities. On the other hand, blast-like cells exhibited an absence of phagocytosis and extremely low oxidative capacity, suggesting that this subpopulation is not directly involved in cell-mediated immune response. The flow cytometry analyses carried out in the present study confirmed that granulocytes are the major hemocyte population involved in cellular defense of C. nippona, and allow for further studies on the impact of environmental stresses or pathogens on this alternative aquaculture species.     

三疣梭子蟹多克隆抗体与4种经济蟹类血细胞交叉反应的研究

采用激光共聚焦扫描显微镜技术、免疫印迹技术和流式细胞术3种方法对鼠抗三疣梭子蟹血细胞多克隆抗体与黄道蟹、日本蟳、中华绒螯蟹、勘察加帝王蟹血细胞的免疫交叉反应进行分析。激光共聚焦扫描显微镜结果表明,该抗体与黄道蟹、日本蟳、勘察加帝王蟹、中华绒螯蟹血细胞均发生了免疫交叉反应,且发生反应的抗原决定簇均位于细胞膜上;流式细胞术测定该抗体与黄道蟹颗粒血细胞和透明血细胞交叉反应阳性率最高,分别为99.45%、98%,与中华绒螯蟹颗粒血细胞和透明血细胞交叉反应阳性率最低,阳性率分别为81.34%、78.56%;免疫印迹结果显示该抗体与日本蟳血细胞结合的蛋白分子量是80、38.5ku;与勘察加帝王蟹血细胞结合的蛋白分子量是80、93ku;与中华绒螯蟹血细胞结合的蛋白分子量是82、41、30ku;与黄道蟹血细胞结合的蛋白分子量是70、43ku。     

圆背角无齿蚌损伤后血细胞的吞噬活性

模拟人工育珠手术对圆背角无齿蚌外套膜进行务后，研究了其血细胞在０～２５ｄ对荧光极毛杆菌的吞噬活性的变化。结果发现，圆背角无齿蚌４种血细胞中有吞噬能力的主要是颗粒细胞和无颗粒细胞。手术蚌血细胞的吞噬活性从手术后１０天起有了显著升高，在第２０天时达到最大，而后开始下降；血清对手术蚌和对照蚌血细胞的吞噬都具有调理作用。     

Morphological, structural, and functional characterization of the haemocytes of the scallop, Argopecten irradians

Argopecten irradians is one of the most important commercial species of Pectinidae family in China. The internal defense system of mollusks consists of circulating haemocytes. In order to characterize the haemocytes of the scallop A. irradians, light and electron microscopical studies were carried out. Four types of haemocytes were recognized: type I small hyalinocytes (2.38 ± 0.08 μm, 30-35%), type II large hyalinocytes (4.41 ± 0.33 μm, 15-20%), type III small granulocytes (4.15 ± 0.26 μm, 20-25%), and type IV large granulocytes (8.26 ± 0.52 μm, 25-30%). Granulocyte types showed smaller N/C ratios than hyalinocytes. The mean haemocyte concentration was about (3.75 ± 0.65) × 10⁷ cells ml^− 1 of haemolymph. Among haemocytes, 44.7% are granular and 55.3% are agranular. These gave a relatively systematic classification scheme for haemocytes of A. irradians. Three types of granules were identified: type I, with high electron-density; type II, with low electron-density; and type III, with a middle level of electron-density, based on TEM studies. Different haemocyte types were not separated with DDGC of Percoll in this study. Both granulocytes and hyalinocytes showed a phagocytic response to the two strains of bacteria, Escherichia coli and RLOs. The phagocytic ability of granulocyte was significantly higher (41-48%) than that of hyalinocyte (9.2-11.2%).     

虾青素对日本沼虾血细胞密度及吞噬活力的影响

从雨生红球藻粉中提取虾青素，以60mg/kg的浓度添加到日本沼虾（Macrobrachium nipponemis）的配合饵料中，在实验室条件下饲养三周，研究虾青素对日本沼虾血淋巴密度及吞噬活力（吞噬百分比和吞噬指教）的影响。结果显示添加虾青素组的日本沼虾不论是血细胞密度还是吞噬活力都显著高于对照组，表明虾膏素可显著提高日本沼虾的免疫力。     

The feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants

An analysis of the immune activity of herbal extract in vivo and in vitro assays was performed to explore the feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants. In vitro experiment was conducted by adding herbal extract into the medium of primary cultured hemocytes and incubating for 24h. In vivo experiment was carried out by feeding shrimp with diets incorporated with herbal extract for 21 days. Challenge test can be used to directly reflect comprehensive immune capacity of organisms. Four immune indexes, total hemocyte counts (THC and lysozyme, phenoloxidase (PO), and phagocytic activity were examined both in vitro and in vivo. The results showed that effects of herbal extract on THC or lysozyme activity were different between in vitro and in vivo. On the contrary, effects of herbal extract on PO activity and phagocytic activity in vitro and in vivo showed a strong correlation. Herbal extract, which enhanced PO activity and phagocytic activity in vitro, had ultimate immune-promoting function in vivo. Therefore, it was feasible to use primary hemocyte culture to screen herbal immunostimulants by determining the effects of herbal immunostimulants on PO and phagocytic activities.     

中国对虾血淋巴凝集素的血凝活性与促噬活性

1999年8月，运用血凝活性测定法分别检测中国对虾血淋巴弹簧集素，血淋巴液、血细胞、肌肉、肠、肝胰腺和甲壳的血凝活力，并运用吖啶橙血细胞吞噬测定法检测血细胞的吞噬活力，同时测定了经注射中国对虾副粘病毒提纯液后血淋巴凝集素血凝活力及血淋巴液抗菌活力、酚氧化酶活力、溶菌活力，结果表明，血淋巴凝集素主要分布在血淋巴液和血细胞中；该凝集素对人（A、B、O型），鸡和兔红细胞及其醛化红细胞的凝集具有一定专一性；对血细胞吞噬异物具有促进功能；注射副粘病毒提纯液后，血淋巴凝集素凝集活力变化和血淋巴液抗菌活力，酚氧化酶活力、溶菌活力变化相一致。