0#柴油对马粪海胆细胞DNA的损伤

如今,国际上石油烃泄漏事故频频发生,防止石油烃对海洋环境造成污染已经追在眉睫.随着海洋温度、光照、潮汐等方面的影响,很大一部分石油烃会沉入海底,对底栖生物造成影响.本文选取生活在海洋底栖环境中、在国际上作为模式生物—马粪海胆作为试验生物,利用单细胞凝胶电泳技术(SCGE),研究了石油烃对马粪海胆DNA的损伤程度.以0#...     

单细胞RNA测序技术在水产养殖动物上的应用

单细胞测序技术是生命科学领域中的革命性技术,单细胞RNA测序技术的出现为动植物的细胞异质性图谱构建、细胞谱系追踪、免疫响应机制解析等研究提供了强有力的工具。近年来,单细胞RNA测序技术在水产养殖动物中也得到了广泛应用。本文就单细胞RNA测序技术的发展、原理及其在水产养殖动物研究中的应用进行综述,并对其目前存在的问题及未来发展趋势进行分析与展望,以期推动该技术在水产养殖动物研究中更广泛的应用,加速水产养殖动物单细胞水平生命活动的调控机制研究进程。     

黄姑鱼精子的超低温冻存及细胞结构损伤的检测

以HBSS溶液为稀释液,DMSO为抗冻剂,0.25 mL麦细管为冻存管,两步降温法超低温冻存黄姑鱼精子,并用单细胞凝胶电泳(SCGE)技术检测了冻精的DNA损伤,荧光双染色流式细胞仪技术(FCM)检测了冻精的细胞膜性结构损伤。结果表明,DMSO质量分数在5%~20%时,冻精的活力与鲜精相比无显著差异;其中DMSO质量分数在10%时,冻精的激活率、运动时间及寿命分别为85.25%±3.95%、(3.23±0.27) min及(3.83±0.33) min。DMSO质量分数在25%、30%时,冻精的活力显著下降。SCGE检测显示,DMSO质量分数在5%~15%时、冻精的DNA损伤与鲜精相比差异不显著,DMSO质量分数为20%、25%、30%时,冻精的DNA损伤明显加重,冻精的DNA损伤与抗冻剂DMSO的质量分数成正相关。FCM检测显示,DMSO质量分数在5%~20%时,冻精中线粒体及细胞膜结构保持完整性的精子比例与鲜精相比无显著差异,DMSO质量分数在25%、30%时,冻精中的线粒体及细胞膜结构保持完整性的精子比例明显下降。分析认为,较高质量分数的DMSO是引起冻精活力下降,DNA、线粒体及细胞膜结构损伤加重的主要原因。     

持续稳定培养单细胞藻技术

持续稳定培养单细胞藻技术曹丽（辽宁省海洋渔业开发中心大连１１６０１３）关键词：培养，单细胞藻，技术随着栉孔扇贝、海湾扇贝、太平洋牡蛎、魁蚶等品种人工育苗技术的不断发展与完善，育苗室的综合利用，已成为共识。这就要求技术人员能够稳定地提供大量的单细胞藻类...     

单细胞藻类培养技术

单细胞藻中含有大量的多聚不饱和脂肪酸（PUFAs）、EPA和DHA,而且多数的单细胞藻具有生长繁殖快、环境适应力强、培养周期短等特点。可实现在人工控制条件下大量培养获得高产的目的,是鱼、虾、贝类等海产品的理想鲜活饵料,藻类培养质量的好坏将直接影响海产品育苗的成败,因此单细胞藻类的培养技术是育苗成功的基础和关键技术。随着人工育苗技术的成熟,单细胞藻类在贝类、虾蟹类、鱼类人工育苗中得到了广泛应用。以下简述一下藻类培养的注意问题：     

几种单细胞藻类高密度保种技术初探

培养高质量高密度的单细胞藻种,是搞好单细胞藻类生产性培养的前提,也是关键性的一环。几年来笔者通过对几种单细胞藻高密度保种的试验,研究探讨了单细胞藻高密度保种的有关技术问题,已获较好效果。新月菱形藻、牟氏角毛藻、湛江叉鞭金藻等保种密度均可达到1000万个细胞/ml以上,高者可达1400万个细胞/ml左右。现将新月菱形藻高密度保种技术报告如下。     

单细胞藻类夏冬两季培育关键技术要点对比

正随着育苗控温设施的普及使用,鱼虾蟹贝全年人工育苗得以实施。单细胞藻类作为贝类、鱼和甲壳类幼体的优质适口饵料,其培育的好坏直接关系到育苗生产成败。单细胞藻在冬夏两季日常管理操作上有诸多不同,其原因在于单细胞藻生长过程中面临的环境不同。夏季高温、多雨,单细胞藻容易下沉、老化、生长菌膜、被原生动物污染。冬季低温,单细胞藻繁殖速度慢,极易发生藻类饵料供应不上。我们根据多年的生产实践经验,对单细胞藻类冬夏两季培育技术要点进     

单细胞藻类的培养方法

单细胞藻类与我们的水产养殖息息相关,不同的单细胞藻类品种及组成使养殖水体表现出不同的颜色.单细胞藻类是鱼、虾、蟹、贝类等的直接或间接饵料;水体中单细胞藻类的优劣决定了鱼、虾、蟹、贝类等生态环境的好坏.在水体中培养单细胞藻类有多种方法,笔者将其总结如下：     

鱼类性别鉴定研究进展

简述了鱼类性别的决定,受遗传、环境因素共同影响;介绍了鱼类性别鉴定方法：表观观测法、组织切片、超声鉴定法、血液生化指标及激素判别法、染色体形态以及染色体带型差异判别法、分子标记法、单细胞测序技术等。分析了各种方法的优缺点及其实际应用范围。提出,今后对于鱼类性别鉴定方法,应着重研究性别特异位点,创新筛选技术,并加大性别分化机制和性别控制的研究力度,进一步开发单细胞测序技术在鱼类性别鉴定中的应用,对于鱼类,尤其是具有明显性别二态性的鱼类研究和渔业发展,具有重要意义。     

单细胞藻类规模化培养技术要点

随着人工育苗技术水平的成熟,单细胞藻类在贝类、虾蟹类、鱼类人工育苗中得到了广泛应用。单细胞藻类不仅为水生动物幼体提供了适口的饵料,而且在一定程度上改善育苗水质。本文就规模化培养单细胞藻类方面概括了几方面的技术要点,以期为同行业者提供借鉴。1培养准备1.1培养器材     

鱼类精子质量评价研究进展

在鱼类人工繁育中,研究者主要关心的是卵子质量,长期以来对精子质量未引起足够重视.而精子质量同样会影响繁育效果的重要因素.鱼类精子质量的评价指标有多种,如精子活力、运动时间、密度、形态、受精率和生理功能等.其中最传统的评价指标是精子活力,其测定方便,能较准确地预测受精率.将精子运动时间和活力综合考虑可更好地反映精子的运动能力.而精子受精率则是精子质量的直接反映,但会受到卵质等因素的影响.质膜完整性、线粒体功能、染色质结构完整性等可体现精子的质量,但测定方法较繁琐.近年来,鱼类精子质量检测技术迅速发展,计算机辅助精子分析(CASA)、流式细胞术(FCM)分析、低渗肿胀(HOS)、单细胞凝胶单泳(SCGE)等技术的建立,使得测定指标更多样、客观、准确.本文逐一介绍了评价精子质量的各种指标,并对各指标的测定方法、测定原理、国内外研究情况进行详细叙述,旨为我国鱼类精子质量评价研究提供背景资料.     

超低温冷冻对俄罗斯鲟精子顶体酶活性及DNA损伤的影响

选取6 ind健康的雄性俄罗斯鲟(Acipenser gueldenstaedti),经人工催产后获得成熟的精子,研究超低温冷冻(-196℃)对俄罗斯鲟精子顶体酶活性及DNA损伤影响。结果显示:俄罗斯鲟鲜精中顶体酶的平均活性为(36.18±2.54)μIU·10-6,经过超低温冷冻后,精子顶体酶活性显著降低,添加抗冻保护液精子中顶体酶活性降至(21.55±0.79)μIU·10-6,未添加抗冻保护液精子中顶体酶活性降至(9.58±1.08)μIU·10-6,且三者间有显著性差异(P0.05)。单细胞凝胶电泳结果表明,俄罗斯鲟鲜精彗星率为(37.33±7.77)%,添加抗冻剂后冻精的彗星率为(63.67±5.13)%,未添加抗冻剂直接冷冻彗星率高达(86.00±3.61)%,三者间有显著差异(P0.05)。用CASP分析软件分析测量彗星拖尾长度(L tail)、彗星尾部DNA的相对含量(Tail DNA)、尾动量(TM)、Olive尾动量(OTM)等各项表征DNA损伤的指标,发现冻精组的各项指标均显著高于鲜精组,未添加抗冻剂直接冷冻组又高于添加抗冻剂组,3组间有显著性差异(P0.05)。本研究结果表明:超低温冷冻能导致精子顶体酶活性下降和DNA损伤,抗冻剂对精子具有保护作用。     

鳜下丘脑神经元细胞培养

为了建立可行、高效的下丘脑神经元细胞技术,本研究通过分离鳜下丘脑组织,用Ⅰ型胶原酶将组织消化成单细胞悬液,并用完全培养液进行培养,在培养的第3、4、5和6天观察细胞的形态。结果显示,培养第3天时细胞贴壁量少,胞体小并且呈单个分布;在培养第4天,细胞的数量显著增多,且具有典型的神经元的形态,胞体饱满;第5天神经元胞体的融合度进一步增大,突起增长增粗,许多分支互相交错连接而形成密集的神经纤维网络;第6天细胞活性减弱,开始凋亡。使用CCK-8法检测细胞活性,结果显示,在培养第5天,细胞活性最高;采用荧光定量PCR(RT-PCR)法和免疫荧光鉴定法对神经元细胞进行鉴定,经RT-PCR检测发现,培养的下丘脑细胞可以表达神经元特异基因noggin,经免疫荧光鉴定,下丘脑神经元细胞纯度为95.9%。研究表明,通过此培养方法能够获得纯度较高的鳜下丘脑神经元细胞,为进一步在细胞水平研究鳜的摄食与能量代谢相关机理奠定基础。     

GnRH and gpcr: laser-captured single cell gene profiling

We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.     

Localization of calcium regulatory hormones in fish

Immunocytochemical localization of hypocalcin, a hypocalcemic factor in the corpuscles of Stannius (CS), in American eels was examined at the light (ABC method) and electron microscopic (protein A-gold technique) levels with the specific antiserum raised against purified rainbow trout hypocalcin. Only type 1 cells in the CS were immunoreactive in the light microscopic immunocytochemistry. At the electron microscopic level, however, hypocalcin immunoreactivity was observed in secretory granules of both type 1 and type 2 cells. Our findings may indicate that type 1 cells are the main source of hypocalcin, but that type 2 cells also produce it, suggesting that the presence of two cell types reflects different physiological conditions of a single cell type, rather than functionally different cell types. In addition, we summarize our recent data on the localization of other calcium regulatory, or putative calcium regulatory, hormones in fish: parathyroid hormone, calcitonin and calcitonin gene-related peptide.     

ONPG单管定量检测法与国标法在检测海水中大肠菌群的比较研究

本实验室根据ONPG显色技术原理建立了大肠菌群单管定量检测技术,即在此基础上利用ONPG单管定量检测方法和国标法对实验室制备的标准菌的菌悬液和水样进行检测,对2种方法的检测结果进行比对.研究结果显示:两种方法对菌悬液、水样的检测结果无统计学差异,ONPG单管定量检测方法检测数据的准确性、精确性和重现性都高于国标法;ON...     

New methods for the primary culture of gill epithelia from freshwater rainbow trout

Gill epithelia from freshwater rainbow trout can be grown in primary culture in Leibovitz's L-15 medium, by seeding freshly isolated gill cells on two successive days, from two different fish, directly onto permeable filter supports (DSI technique). This preparation allows the measurement of transepithelial resistance (TER) and exposure of the apical surface to freshwater, as in vivo. New culture methods were developed and evaluated, using TER as an indicator of epithelial integrity, in an effort to improve the utility of the preparation for proteomic and toxicological research. TER was not related to cell density or protein content in DSI epithelia. To eliminate bovine proteins, the 5% foetal bovine serum (FBS) normally required for epithelial development was replaced with trout plasma. While previously frozen trout plasma proved toxic, freshly collected heparinized plasma, provided by chronically cannulated adult trout, was not. The use of 5% fresh trout plasma supported a TER development curve identical to that with 5% FBS, a useful advance for proteomic research because foreign (bovine) proteins are eliminated. However, 10% plasma reduced TER development, and 100% plasma abolished it. The inhibitory effect on TER of high plasma levels was seen only early in epithelial development, and was exerted from the apical side, likely an effect on tight junction formation. Mature plasma-supplemented preparations mounted a TER rise in response to apical freshwater exposure comparable to that of FBS-supplemented epithelia. Yolk-sac fry extract was inhibitory to TER development, even in the presence of 5% FBS. Transfer of mature epithelia from 18 °C to 4 °C maintained stable TER and extended the useable lifespan by at least ten days, thereby facilitating storage of preparations for toxicity testing. A new method of growing epithelia, involving only a single seeding of cells from a single fish, directly onto filter inserts (SDSI technique), provided mature epithelia with much lower TER, a smaller TER response to apical freshwater, and lower cell density and protein content than DSI epithelia. These SDSI epithelia offer the advantage of multiple preparations grown directly from unique individuals for in vitro toxicity testing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of a multiplex RT-PCR assay for simultaneous detection of the major viruses that affect rainbow trout (Oncorhynchus mykiss)

The major viral diseases that affect rainbow trout (Oncorhynchus mykiss) are viral haemorrhagic septicaemia, infectious haematopoietic necrosis, infectious pancreatic necrosis and sleeping disease. In the presented study, we developed a multiplex RT-PCR (mRT-PCR) assay for the simultaneous detection of these four rainbow trout viruses in a single assay. The choice of primers was carried out based on the expected size of the fragments, the temperature and time required for the amplification, and the specificity for the target sequence. Firstly, the method was optimised using reference strains of viral haemorrhagic septicaemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV) and sleeping disease virus (SDV) cultivated with permissive cell culture lines; subsequently, the method was used for the identification of these viral infections in rainbow trout samples. Twenty-two samples of rainbow trout, clinically suspected of having viruses, were analysed by the developed method to detect the presence of the four viruses, by directly analysing the animal tissues. The mRT-PCR method was able to efficiently detect the viral RNA in infected cell culture supernatants and in tissue samples, highlighting the presence of single infections as well as co-infections in rainbow trout samples. VHSV/SDV and IHNV/SDV co-infections were demonstrated for the first time in rainbow trout. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.     

枝管藻细胞壁内、外多糖的细胞化学定位研究

为进行经济海藻枝管藻细胞壁多糖和胞外多糖的超微定位,本文以钌红电镜细胞化学方法制备枝管藻透射电镜样品。结果表明,钌红染色方法不仅比常规电镜制样方法能够更完整地显示海藻细胞壁的超微结构,而且使枝管藻多糖在细胞壁内、外的分布在透射电镜下直观可见:细胞壁内的多糖物质主要呈多层紧密排列的微纤维状,细胞表面的多糖物质则呈松散的网状。本结果为研究海藻细胞壁的超微结构和海藻吸附重金属的作用部位提供了技术支持。     

Identification of marine algicidal Flavobacterium sp. 5 N-3 using multiple probes and whole-cell hybridization

ABSTRACT: We designed five 16S rRNA-targeted oligonucleotide probes (Sp probes) specific for Flavobacterium sp. 5 N-3, which inhibits the growth of red tide phytoplankton Gymnodinium mikimotoi (Dinophyceae). These probes were evaluated by whole-cell hybridization against 5 N-3 cells incubated under laboratory conditions. The fluorescence signal from the cell detected with Sp probe mix5, a mixture of the five probes, was 8.4-fold higher than that obtained with only one Sp probe (Sp01RF). The signal obtained by this method was strong enough to recognize 5 N-3 cells and count them under the epifluorescence microscope, while the signal was often undetectable when a single probe was used. Fluorescence intensities of cells at stationary phases and of 'starved' cells in sterile seawater using Sp probe mix5 were low but still sufficient for enumeration. These Sp probes did not hybridize with 11 strains from the Cytophaga/Flavobacteria/Bacteroides phylum and did react with strain 5 N-3 following whole-cell hybridization. These results show that 5 N-3 cells cultivated under our laboratory conditions can be detected by whole-cell hybridization with the five designed probes. These data also suggest that this technique may be useful for detection of an algicidal bacterium 5 N-3 in the natural environment.