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。     

橄榄蛏蚌血细胞形态及吞噬能力的初步研究

对橄榄蛏蚌(Solenaia oleivora)血细胞的形态特征和吞噬作用进行了初步研究。基于瑞氏染色后血细胞的形态、颜色、大小等特征,可将橄榄蛏蚌的血细胞分为大颗粒细胞、小颗粒细胞、透明细胞、类淋巴细胞共4种类型。透明细胞的直径最大,小颗粒细胞次之,类淋巴细胞最小;透明细胞的胞核直径最大,小颗粒细胞次之,大颗粒细胞最小。类淋巴细胞的核质比最大(0.67),其他3种细胞的核质比为0.37～0.48。4种血细胞所占比例以小颗粒细胞最高(43.6%),透明细胞次之(36.6%),类淋巴细胞最低(7.0%)。橄榄蛏蚌平均血细胞浓度为(4.21±1.51)×105个/mL,个体的血细胞浓度与体重(或壳长)不相关(P>0.05),不同体重组(或壳长组)的平均血细胞浓度无显著差异(P>0.05)。在37℃体外吞噬试验中,橄榄蛏蚌血细胞对金黄色葡萄球菌的吞噬比例为(43.6±7.0)%,吞噬指数为(0.743±0.145)。4种血细胞中,小颗粒细胞的吞噬比例最大(0.288±0.061),类淋巴细胞不具吞噬能力。     

亚硝酸盐对凡纳滨对虾血细胞毒性及p53基因表达的影响

随着水产动物集约化养殖的迅猛发展,水质污染问题日益加剧,环境中亚硝酸盐的含量不断上升,成为水产养殖中诱发爆发性疾病的重要环境因子。为探讨亚硝酸盐对凡纳滨对虾（Litopenaeus vannamei）血细胞的毒性以及 p53 mRNA 的影响,通过预试验以及前期资料获取亚硝酸盐对凡纳滨对虾的毒性效应,选取0 mg／L 对照组和20 mg／L 浓度组,用流式细胞术检测血细胞活性氧（ROS）、一氧化氮（NO）含量和非特异性酯酶活性,运用荧光定量 PCR 技术检测 p53基因表达量变化。结果显示,在亚硝酸盐应激12 h 后,对虾血细胞的 NO 含量与对照组相比有显著升高（P ＜0．05）,在应激24、48、72 h 后有极显著的升高（P ＜0．01）;ROS 含量在应激12、48、72 h 时有极显著升高（P ＜0．01）,在应激24 h 时与对照组相比有显著的升高（P ＜0．05）。非特异性酯酶活性在应激24、48、72 h 显著下降（P ＜0．05）,p53的表达水平在应激48 h 和72 h 后显著升高（P ＜0．05）。研究表明,亚硝酸盐应激诱导对虾血细胞产生了过量的 NO 和 ROS,造成氧化胁迫作用,诱导凋亡相关基因的表达,最终导致细胞凋亡。     

中华绒螯蟹的血细胞组成、分类及免疫学功能

为了解中华绒螯蟹血细胞组成、分类及其在免疫应答过程中起到的重要作用,本研究通过细胞化学和细胞酶学分析,并结合细胞形态观察,对中华绒螯蟹血细胞的组成、分类进行了研究。同时,通过人工感染嗜水气单胞菌后血液的总血细胞数(THC)和不同类型血细胞数(DHC)的变化,研究不同类型血细胞在免疫应答过程中所发挥的作用。结果显示,依据本实验分类方法,中华绒螯蟹血细胞可以分为4类:大颗粒细胞(G)、中间型颗粒细胞(IG)、小颗粒细胞(SG)和透明细胞(H)。其中小颗粒细胞数量最多,约占33.54%±0.98%,中间型颗粒细胞最少,仅占15.31%±2.01%。4种类型细胞中均含有多糖成分而不含脂质,只有大颗粒细胞在酸性磷酸酶、碱性磷酸酶、β-葡萄糖醛酸酶以及酚氧化酶染色中发现阳性反应。此外,嗜水气单胞菌感染后中华绒螯蟹总血细胞数在3 h后明显升高,在6 h达到峰值,约9.57×106个/m L,并显著高于未处理组和生理组;在感染过程中,大颗粒细胞数量明显下降而透明细胞数量明显上升,这种现象在6 h时达到顶峰并随着时间延长而逐渐恢复至正常水平。研究表明,在中华绒螯蟹非特异性免疫应答过程中,透明细胞主要通过大量增殖执行吞噬功能来参与免疫应答,而大颗粒细胞主要通过裂解释放胞质中所含免疫相关酶参与免疫反应。其中,各类型细胞之间可能存在相互转化作用,而中间型颗粒细胞为这种转化中的过渡类型。     

皱纹盘鲍血细胞的亚显微结构及分类研究

通过对皱纹盘鲍血细胞的超微结构的观察，将其血细胞分成5种类型：大颗粒细胞、小颗粒细胞、特殊颗粒细胞、透明细胞和淋巴样细胞。根据胞核的形态和胞质中细胞器的结构特征，研究认为大、小颗粒细胞是同一类细胞的不同发育阶段，而特殊细胞则是大、小颗粒细胞的原始细胞；透明细胞和淋巴样细胞是两类完全分化了的细胞。     

红螯螯虾血细胞分类、结构与免疫特性研究

血细胞在甲壳动物免疫过程中起重要作用,探讨红螯螯虾(Cherax quadricarinatus)不同类型血细胞结构与免疫特性有助于其病害防治。应用流式细胞术(FCM),根据前向角散射光(FSC)和侧向角散射光(SSC)的强度差异对血细胞进行分类,利用特异性荧光染料进行标记,分析血细胞总数(THC)、线粒体数量、溶酶体数量、吞噬活力、活性氧(ROS)含量、一氧化氮(NO)含量和非特异性酯酶活力。结果显示,应用FCM可以区分透明细胞(HC)、半颗粒细胞(SGC)和颗粒细胞(GC)三类血细胞,其占比分别为9.82%、61.11%和25.24%;红螯螯虾平均血细胞总数(THC)为(8.43±0.87)×10~6个/mL;GC含有最多数量的线粒体和溶酶体,HC中含量最少;HC、SGC和GC的吞噬率分别为2.54%、14.45%和6.98%,SGC吞噬活力最强,HC最弱;HC、SGC和GC的活性氧(ROS)含量分别为7.80、45.95和134.69 AU,GC的ROS含量最高,HC最低;HC、SGC和GC的一氧化氮(NO)含量分别为8.20、79.78和344.31 AU,GC的NO含量最高,HC最低;HC、SGC和GC的非特异性酯酶活力分别为86.59、121.84和236.91AU,GC的酯酶活力最高,HC最低。研究表明,红螯螯虾三类血细胞在形态结构、数量及免疫特性上均存在差异,SGC的数量最多、吞噬活力最强,而GC含有最多与能量供应、免疫防御相关的细胞器,并拥有最强的氧化活力和酯酶活力,表明GC和SGC是红螯螯虾免疫防御过程中发挥主要作用的血细胞类型。     

菲律宾蛤仔、中国蛤蜊、文蛤和紫石房蛤血细胞的分类研究

采用流式细胞仪对菲律宾蛤仔、中国蛤蜊、文蛤和紫石房蛤4种双壳贝类的血细胞分类进行了比较研究。通过比较4种贝类的前向角散射光(FSC)和侧向角散射光(SSC)双参数点图以及FSC和SSC单参数直方图,发现4种贝类的血细胞均可根据其细胞质中是否有颗粒分为透明细胞和颗粒细胞两种基本类型,中国蛤蜊和紫石房蛤的颗粒细胞又可分为小颗粒细胞和大颗粒细胞两个亚群,菲律宾蛤仔的颗粒细胞又可以区分为小颗粒细胞、中颗粒细胞和大颗粒细胞3个亚群,而文蛤只有颗粒细胞,通过统计分析双参数点图中的各细胞亚群,4种贝类血细胞亚群的含量存在一定差异。     

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%。     

流式细胞术与显微观察对罗氏沼虾血细胞的比较研究

应用流式细胞术和显微观察对罗氏沼虾（Macrobrachium rosenbergii）血细胞的分类分群和各类细胞组成比例进行研究，比较2种方法的优缺点。应用显微观察，根据细胞颗粒的有无和大小以及细胞染色情况，可把罗氏沼虾血细胞分为透明细胞、小颗粒细胞和大颗粒细胞。根据其血细胞的前向角散射光（FSC）和侧向角散射光（SSC）特性的不同，也将血细胞分为3个亚群：透明细胞、小颗粒细胞和大颗粒细胞。检测各种细胞所占的比例，2种方法测得比例均为：小颗粒细胞〉大颗粒细胞〉透明细胞；2方法的结果存在显著差异（P〈0．05），但相关性极显著（P〈0．01）。利用流式细胞术可更快捷、准确，减少人为误差，显微观察可对细胞内部结构进行更深入的分析，并可进行细胞实际大小和细胞密度的测定。2种方法互补不足，可更快速、准确、全面地进行细胞基础特征的研究。     

硫化物胁迫对凡纳滨对虾血细胞的毒性影响

硫化物是水产养殖过程中常见的水体污染物之一。为探讨水体硫化物胁迫对虾类血细胞的毒性影响,以不同浓度(0.5 mg/L,2.0 mg/L)的硫化物对凡纳滨对虾(Litopenaeus vannamei)进行胁迫,于胁迫后6、12、24和48 h取血淋巴,应用流式细胞术测定血细胞的总数(THC)、活性氧(ROS)含量、一氧化氮(NO)含量以及凋亡率。结果显示,经0.5 mg/L硫化物胁迫48 h后,对虾THC显著下降至11.78×10~6个/mL,为对照组的72.7%(P0.05),ROS含量为对照组的225.2%(P0.05),血细胞凋亡率显著上升至7.42%(P0.05);经2.0 mg/L硫化物胁迫6 h开始,对虾THC呈现显著的下降(P0.05),血细胞ROS含量和凋亡率显著提高(P0.05);表明硫化物胁迫刺激对虾血细胞产生大量ROS,诱导血细胞凋亡,从而导致THC下降,这一过程可能是硫化物导致虾类细胞免疫下降的重要机制;随着硫化物浓度的升高,其细胞毒性作用明显提高。血细胞NO含量在2.0 mg/L硫化物胁迫12和48 h时显著升高(P0.05),推测NO可能在硫化物胁迫防御调控中起着信号因子的作用。     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.     

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%).     

Tanner crab Chionocetes bairdi Rathbun haemocyte classification and an evaluation of using differential counts to measure infection with a fungal disease

Abstract. The circulating haemocytes of the tanner crab, Chionocetes bairdi , were studied and classified as hyalinocytes, intermediate granulocytes and eosinophilic granulocytes. Differential haemocyte counts of normal crabs and crabs infected with an apparently pathogenic unnamed ascomycete fungus revealed a shift in the hyalinocyte-granulocyte ratio in infected crabs. There was a highly significant increase in the percentage of granulocytes and a corresponding decrease in hyalinocytes in infected crabs. This shift was thought to be caused by a marked increase in the relative number of eosinophilic gianulocytes. There was also a highly significant statistical relationship between the severity of the internal fungus infection, established by histopathological analysis of the major organ systems, and the increased number of circulating eosinophilic granulocytes. The results of the study indicate that differential haemocyte counts may be a valuable tool in monitoring the health of C. bairdi populations.     

Differences in uptake and killing of pathogenic and non‐pathogenic bacteria by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.     

饥饿胁迫对虾夷扇贝几种免疫因子的影响

在(17±1)℃条件下,研究了壳长(8.83±0.52)cm的虾夷扇贝在不同饥饿时间(5、10、15、20、30 d)处理后,血液中血细胞总数、血细胞吞噬水平、酸性磷酸酶、过氧化物歧化酶的变化。结果表明,扇贝的血细胞总数随着饥饿时间的延长逐渐降低,饥饿20 d以上的扇贝血细胞数明显降低,与投喂组差异极显著(P<0.01),扇贝血细胞吞噬率和酸性磷酸酶活性呈现出先小幅上升后下降的趋势,饥饿253、0 d与投喂组差异显著(P<0.05),饥饿253、0 d的扇贝过氧化物歧化酶的活性有所升高,饥饿30d,与投喂组差异显著(P<0.05)。试验表明,饥饿超过20 d,扇贝的免疫系统受到较为严重的破坏。