中国对虾类淋巴组织培养中 细胞异常的病理研究

在对养殖中国对虾和海捕中国对虾的类淋巴 器官进行组织培养时,发现迁出的细胞空泡化、细胞内颗粒较多。培养一周后由养殖中国对 虾类淋巴组织迁出的细胞开始大量脱落,而由海捕中国对虾类淋巴组织培养出的细胞可生长 30d左右。对未经培养的类淋巴组织及培养的类淋巴组织进行透射电镜观察,发现在养殖中 国对虾类淋巴组织的细胞中除了有球形病毒及球状和多面体状病毒包涵体外,在细胞质中还 含有大量类立克次氏体样原核生物,而且每个细胞中的类立克次氏体均由一单层膜包在一起 ;在海捕中国对虾类淋巴组织的细胞中也发现有球形病毒及球状病毒包涵体,另外还在少量 细胞中发现有类立克次氏体样原核生物存在。感染病毒的组织细胞呈现空泡化、核膜肿胀破 裂、核质固缩、高尔基体肿胀、线粒体嵴消失等病理变化。感染有类立克次氏体样原核生物 的细胞不同程度地呈现出细胞器消失、核染色质固缩等病理结构。     

磺胺甲基唑和恩诺沙星在日本囊对虾体内的药代动力学研究

为了研究磺胺甲基唑(Sulfamethoxazole, 简称SMZ)和恩诺沙星(Enrofloxacin, 简称ERFX)在日本囊对虾(Marsupenaeus japonicus)体内的药物代谢动力学特点, 在水温21℃±0.5℃, 盐度29.91 的条件下, 应用萃取、液相测定和反相高效液相色谱法(RP-HPLC)对药物平均回收率、主要动力学参数及在3种组织中的半衰期进行了测定, 检测数据经药物代谢动力学软件3p97 进行研究。结果表明, SMZ 和ERFX 在对虾肝脏、肌肉和血淋巴的平均回收率分别为90.09％, 88.92％, 84.84％和89.14％, 86.04％,91.77％。药物代谢动力学分析表明, SMZ 单次腹部肌肉注射日本囊对虾, 其肝脏药时数据符合二室模型, 肌肉和血淋巴药时数据符合一室模型,半衰期为19.1284, 4.5799, 9.1855 h。ERFX 单次腹部肌肉注射日本囊对虾, 其肌肉药时数据符合二室模型, 肝脏和血淋巴药时数据符合一室模型。半衰期分别为15.16, 16.83, 17.19 h。通过两种药物的代谢特征比较分析表明, SMZ 在肌肉中吸收较ERFX 快, ERFX在血淋巴和肝脏中吸收较SMZ 快。根据SMZ 和ERFX 在21℃±0.5℃的条件下的主要动力学参数及3种组织内消除半衰期, 建议SMZ 在日本囊对虾的休药期不少于10 d, ERFX 的休药期不少于12 d。     

大泷六线鱼脾脏细胞体外培养的研究

应用植块法,比较大泷六线鱼(Hexagrammos otakii)脾脏细胞在2种基础培养基和3个胎牛血清浓度下的体外培养效果。根据细胞的迁出、贴壁、细胞种类以及生长状况,得出大泷六线鱼脾脏细胞适宜的体外培养体系为:MEM+15%胎牛血清、pH7.2~7.4、20℃;培养产物由6种细胞组成,其中C型细胞培养120 d以上依然状态良好,并进行了传代培养。培养启动12 d后,C型细胞层上出现造血灶,造血灶细胞最长可以维持50 d以上。本研究为大泷六线鱼脾脏细胞系的构建和脾脏功能的研究奠定了基础。     

寿光县对虾养殖环境中微生物数量分布的试验报告

本文主要介绍了1988年寿光县对虾养殖期间(5～9月)水环境和底泥环境中微生物种群的变化。据不同的地理位置,选择不同的养殖虾池;分别将水样和底泥环境中的弧菌、异养菌,及硫酸盐还原菌进行培养、分离计数。据对虾养殖期5个月的数据分析,不同的养殖虾池有不同的菌数波动范围。底泥与水环境中的菌数分布各有不同。异养菌在水样中的分布最高值为736×10~2个/ml,最低值为268.1×10~2个/ml;湿泥样中的最高值为372.7×10~4个/g最低值为125.9×10~4个/g。弧菌在水样中的最高值为12.33×10~2个/ml,最低值为0.5×10~4个/ml。据综合资料分析,对虾养殖池中的各种细菌分布,底泥高于水样,高温季节高于低温季节。底泥和水环境中有机物的含量与细菌数的分布成正比。底泥中硫酸盐还原菌的数量,是底泥环境中有机物污染程度的标志之一。根据硫酸盐还原菌的数量分布,可以有效地控制有机质(如对虾饵料)的投放量等。     

锦鲤组织细胞体外培养的初步研究

为建立来源于锦鲤的细胞系,本文采用组织块法,对来源于锦鲤(Cyprinus carpio)鳍条、吻端、肌肉、心脏、鳔、肠道、卵巢等组织的细胞进行原代培养和传代培养.在25℃下,添加20％胎牛血清、0.2μg/mL表皮生长因子(EGF)和25ng/mL成纤维生长因子(FGF)的L-15培养基中进行培养.结果显示,鳍条、心脏、鳔、吻端、肌肉、肠道、卵巢分别在原代培养第3、6、12、13、15、3、3天,有细胞从组织块迁出;对长至单层的鳍条、心脏、肌肉细胞进行传代培养,鳍条细胞已传至第39代,心脏和肌肉细胞分别传到第2代和第4代;鳍条、心脏和肌肉细胞分别呈现上皮细胞样、上皮细胞样和上皮细胞样与成纤维状混合型.第6代锦鲤鳍细胞的染色体计数,结果显示,细胞染色体数目分布范围为55～154条,2n=100.病毒敏感性实验,发现锦鲤鳍条细胞对草鱼出血病病毒(GCRV)和鲤春病毒血症病毒(SVCV)都敏感,且在24h内出现细胞病变(CPE);但对斑点叉尾鮰病毒(CCV)不敏感.锦鲤鳍条细胞系的建立为后期建立更多的锦鲤细胞系和鱼类病毒研究奠定了基础.     

日本七鳃鳗外周血细胞显微结构及类淋巴细胞体外培养

以日本七鳃鳗(Lampetra japonica)为研究对象,观察其外周血细胞显微结构和探讨类淋巴细胞原代培养条件。采用Wright氏和Giemsa氏染色法对日本七鳃鳗外周血液有形成分进行显微观察,可鉴别出红细胞、淋巴细胞、中性粒细胞、单核细胞、嗜酸性粒细胞;未发现嗜碱性粒细胞。通过Ficoll密度梯度离心法和流式细胞仪分选获得高纯度的七鳃鳗类淋巴细胞。经条件优化后确定最适培养液L15+20%FBS(5%七鳃鳗血清)+0.46%NaCl,在18℃,pH 6.8-7.0的条件下对日本七鳃鳗类淋巴细胞进行体外培养,大多数类淋巴细胞半贴壁生长,细胞状态较好,最长可以存活近一个月。类淋巴细胞的原代培养为日本七鳃鳗细胞体外培养的深入研究提供了实验依据。     

中国对虾淋巴组织的长期原代培养和化学诱变初探

以中国对虾为实验材料 ,研究影响对虾淋巴组织原代培养的一些因素。研究中发现 ,用75%的乙醇浸泡对虾就可以达到淋巴组织培养无菌的要求 ,当组织块的接种密度为 2 4 0～ 2 60块( 2 5cm2 ) -1时最适。当原代细胞培养 30 d左右 ,细胞会因接触抑制而停止生长 ,当接触抑制消除后 ,细胞仍可继续生长。另外 ,还进行了以 N-甲基 - N′-亚硝基胍 ( MNNG)对原代细胞进行诱变的尝试发现诱变后有的细胞呈现出转化细胞的形态 ,并形成了转化灶     

实验感染白斑杆状病毒(WSBV)的斑节对虾血液病理学研究

用光镜观察经人工感染白斑杆状病毒 (White Spot Baculovirus,WSBV) 后患典型白斑病的斑节对虾 Penaeus monodon 的血淋巴涂片,研究了WSBV导致的淋巴细胞数量及结构异常;通过检测感染WSBV后患病虾与健康虾的血清主要生化指标,首次揭示了两者的主要差异及WSBV在宿主体内增殖的时间过程.结果表明:患病虾淋巴细胞明显减少,无颗粒细胞难以找到,颗粒细胞和小颗粒细胞分别为健康虾的11.7%和4%;颗粒细胞内出现深染区、空白区或异样突起.血涂片上偶尔发现有大型固定性吞噬细胞.在患病虾血清主要生化指标中,Ca2+含量比健康虾显著升高,患病虾为15.93mmol·L-1,健康虾为10.71mmol·L-1;相反,患病虾血清中总蛋白(Tp)、总磷(PB)及乳酸脱氢酶(LDH)均比健康虾个体低,3者含量依次为:52.07g·L-1,0.34mmol·L-1和35.27IU·L-1,在健康虾个体血清中为:66.29g·L-1,0.66mmol·L-1及82.36IU·L-1;而K+,Mg2+含量无显著差异.血清中Ca2+,PB及TP的含量可以作为斑节对虾感染WSBV后出现白斑病的血清生化特征.斑节对虾感染WSBV后不同时间内血清主要生化指标显示其增殖的时间顺序可能为:24h内主要进行核酸复制,24-48h主要进行囊膜蛋白质的合成,病毒从入侵宿主细胞到从细胞内释放的过程约需48h.     

四脊滑螯虾造血组织细胞培养条件的优化

稳定的体外细胞培养体系,可以为基因功能和宿主 病原相互作用研究提供平台。本研究通过对四脊滑螯虾(Cherax quadricarinatus)造血组织(hematopoietic tissue, HPT)细胞的体外培养条件进行优化,以期获得状态稳定、活力良好的造血组织细胞培养物。我们从四脊滑螯虾中分离提取造血组织细胞后,通过对培养基种类(L 15、Express Five、DMEM),谷氨酰胺浓度(0、1%、5%、10%),胎牛血清浓度(0、1%、5%、10%),以及体外培养温度(20、27 ℃)等培养条件进行优化。细胞形态观察和细胞活力分析结果表明,HPT细胞在添加1%青霉素 链霉素、10%L 谷氨酰胺、1%胎牛血清的L 15培养基中状态最佳,在20 ℃培养温度下能存活30 d左右。这些培养条件将有利于四脊滑螯虾造血组织细胞的体外培养。     

褐点石斑鱼三种组织细胞系的建立

为建立褐点石斑鱼鳍、心脏和鳔组织细胞系,本文利用不同培养液和培养温度对褐点石斑鱼鳍、心脏和鳔组织细胞进行了原代培养和传代培养。实验结果显示,在24℃培养于含有羧甲基壳寡糖、碱性成纤维样生长因子、I型胰岛素样生长因子及20%胎牛血清的DMEM/F12培养液(pH=7.2)中的3种细胞,均为成纤维样细胞,其生长分裂状态最佳,可以持续稳定传代。第60代褐点石斑鱼鳍、心脏和鳔细胞的群体倍增时间分别为50.6 h、40.3 h和43.3 h,其特征性染色体数目均为48条。目前鳍、心脏和鳔细胞已分别传至第90代、第70代和第75代,已成功建立了3种细胞的连续性细胞系,为鱼类病毒与宿主细胞相互作用机制等鱼类病毒学基础研究,以及鱼类病毒的分离、鉴定、繁殖及病毒疫苗研制等奠定了基础。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.