免疫磁珠法分离鼻咽癌基质的T淋巴细胞

肿瘤内环境与肿瘤的发生密切相关.肿瘤细胞周围的组织在癌变发生时不会是一个沉默的旁观者,可能在肿瘤的发生和发展中扮演十分重要的角色.本研究分别采用不同的磁珠分选技术分离T淋巴细胞.采用CK LMP1,CD105和成纤维细胞表面蛋白,结合全血总T细胞试剂盒,间接法分离鼻咽癌基质的T淋巴细胞;采用CD3直接磁分选法分离鼻咽癌基质的T淋巴细胞,然后用免疫组化法鉴定分选的效果和细胞的质量.结果表明,免疫组化显示间接磁分选法分离出来的T淋巴细胞不能完全去除肿瘤细胞,RNA的质量不佳;而直接磁分选分离出来的T淋巴细胞为纯净的T淋巴细胞,而且RNA的质量良好.提示直接磁分选技术是分离鼻咽癌基质T淋巴细胞的首选方法.     

Lymphocyte culture for chromosome preparation

A technique is described using separated peripheral blood lymphocytes from fish which yields cultures giving good chromosome spreads suitable for convential karyotyping and the subsequent application of banding techniques.     

Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes

Optimum procedures for fish handling and sample processing for use when employing haematological parameters as health indicators in turbot, Psetta maxima (L.), have been established. We found thrombocytes to be the most abundant blood cell, representing approximately 52% of circulating leucocytes (lymphocytes, 40.8%; granulocytes, 5.6%; monocytes, 1.6%; total number of leucocytes=1.3 × 10⁵ ml⁻¹; packed cell volume=22.7%). The light- and electron-microscopical characteristics of these cell types are described, together with their cytochemical properties using Sudan Black B, Periodic Acid Schiff, Non-specific Esterase, and Acid and Alkaline Phosphatase. Turbot thrombocytes showed a high degree of shape alterations when observed in live preparations using phase contrast microscopy, while ultrastructural observations following the in vitro uptake of carbon particles supported an active process of phagocytosis by the thrombocytes, rather than passive entrapment. The lymphocytes of turbot are structurally similar to mammalian lymphocytes with the highest nuclear:cytoplasmic ratio of all the leucocytes observed. Small lymphocytes predominated, large lymphocytes forming less than 1% of the total white blood cell population. The most frequent granulocyte type was a neutrophil-like cell with an eccentric nucleus, only rarely seen in segmented form. In vitro uptake of carbon particles by granulocytes was not observed under the conditions of the experiment, although turbot granulocytes are capable of phagocytosis under different circumstances. These are discussed, along with other physiological and technical factors which can influence the blood parameter findings in fish.     

Utilization of shrimp industry waste in the formulation of tilapia (Oreochromis niloticus Linnaeus) feed

A rapid expansion of fisheries is demanding an adequate supply of efficient, nutritious and inexpensive fish feed, because feed contributes highly to the cost of fish production. Shrimp head, a waste product from the shrimp export industry qualifies as an economical, abundant and good quality protein source for fish feeds. In the present work, shrimp head silage powder, which contained approximately 40% protein, was used as a substitute for fish flour. Four feeds, in the form of pellets, were prepared by substituting shrimp head silage for fish flour at 0%, 33.3%, 66.6% and 100% dietary levels. Other ingredients such as corn, soy, bovine blood, cassava and corn cob flours, soy oil, vitamin premix, salt, and other components also were used in the formulation. A commercial fish feed was used as the control. The proximate composition of these feeds did not differ significantly at p>0.05, except for the protein content of the control feed, which was about 30.6% versus 35.4-36.9% protein in the other diets. No significant differences (p>0.05 level) in weight and length of juveniles fed with the different feeds during a period of 60 days were observed. In all cases, an excellent correlation (0.9950-0.9996) between weight and length of juveniles was observed. No significant difference in growth of juveniles fed on R1, R2, R3, or R4, or the control feed, was observed. Similarly, the proximate analyses of the flesh of juveniles did not present significant differences (p>0.05). The result of the study indicates that the shrimp head silage could replace fish flour as an ingredient in tilapia feed with economic advantages and without sacrificing the quality of the feed.     

Characterization of blood cells and hematological parameters in Cichlasoma dimerus (Teleostei, Perciformes)

The aim of the present study was to obtain a basic knowledge of the hematology of Cichlasoma dimerus. The morphological features of blood cells were described according to the observations made by light and electron microscopy. Erythrocytes, thrombocytes and four types of leucocytes: lymphocytes, monocytes, heterophils and eosinophils, were distinguished and characterized. Thrombocytes are the most abundant blood cells after erythrocytes and are recognized easily from lymphocytes by morphological features and size. Heterophils and eosinophils are PAS positive. Hematological indices (RBC, WBC, PCV, Hb, MCV, MCH, MCHC and leucocyte differential count) were measured in one blood sample from 30 adult fish captured in Esteros del Riachuelo, Corrientes, Argentina (27 degrees 25'S, 58 degrees 15'W). The reference interval and the mean were determined for each hematological parameter evaluated. Contrary to other species, the percent of heterophils was found to be high in relation to the percent of lymphocytes. Low lymphocyte counts occurred in C. dimerus, as compared to these found in other fishes. Compared to most teleosts, this species has similar mean values for PCV and Hb and slightly higher for RBC. Statistical analysis revealed that differences in hematological parameters between male and female fish were not significant.     

大川河鱼类区系及其群落生态结构的研究

鱼类区系及其群落生态结构,在理论和实践上都有十分重要的意义。但是,关于鱼类区系的生态结构问题,还未见详细报道。本文通过对大川河鱼类区系及其群落生态结构的分析,探讨鱼类区系及其群落生态结构的规律。     

Comparison of lymphocyte subpopulations in arterial and venous blood of the rat

Lymphocyte subpopulation as well as other hematological properties were compared between arterial and venous bloods of Wistar-Imamichi rats. Lymphocyte subsets were defined with four monoclonal antibodies which were specific to the respective cell surface glycoproteins. Using these monoclonal antibodies, subsets of B lymphocytes, T lymphocytes, helper T lymphocytes, and suppressor and cytotoxic T lymphocytes in the peripheral lymphocytes were identified. The blood samples were taken from aorta abdominalis and venae cava caudalis. The population of these subsets were enumerated by a laser flow-cytometry system. The result showed that there was no significant difference in hematological properties between the arterial and venous blood except in leukocyte count and hemoglobin concentration. The difference in leukocyte counts was thought to depend mainly on the fluctuation of the lymphocyte counts. However, no significant difference was recognized in the proportion of positive cells to each monoclonal antibody. It was concluded that the difference in leukocyte counts found between the arterial and venous bloods of the Wistar-Imamichi rat did not produce any effects on the proportion of the subpopulation in the peripheral lymphocytes, and the lymphocyte subpopulations in both arterial and venous bloods were substantially equivalent to each other.     

人淋巴细胞的体外抗体诱发及其在杂交瘤中的应用

Sources of immunized lymphocytes constitute one of the main obstacles in the production of human monoclonal antibodies. We tried to get them through in vitro immunization. Cells from excised tonsils or trauma spleens were used for the induction of antibody responses in vitro. Antibodies to different antigens including sheep red blood cells, ovalbumin, tetanus toxoid, and hepatitis B surface antigens were induced in 7-14 days' cultures. Taking tetanus toxoid as antigen, we analysed the various factors required for antibody induction with statistics analysis, which included cell separation method, T cell conditioned medium, antigen dosage, serum content, and concentration of mitogen PWM and LPS. The results showed: (1) The cell separation method influenced the antibody production significantly in comparison with other factors. It signified that immune cells' combination was the most influential factor. (2) Serum also constituted quite important influencing factor especially in the later period of culture. However, it did not make much differences if it attended 10% or so. The antigens and mitogens tended to be used at low concentration. (3) Due to the significant variation among individuals and among different antigens, it is suggested to set up the culture system with some flexibility so as to adapt to the variation in cells and antigens from different sources. The present culture system we use includes nylon wall column separation of cells, suitable range of antigens (three doses instead of one), and either 10% T cell conditioned medium or a mixture of 1 microgram PWM/ ml + 0.1 microgram LPS/ml. The human B lymphocytes stimulated in vitro with tetanus toxoid were used for the construction of human hybridomas.     

Effect of bacterial infection on the lipid composition of carp blood lymphocytes

The initial stage of bacterial infection is characterized by an increase in the level of total lipids and polyenoic fatty acids in membrane phospholipids of blood lymphocytes of carp (Cyprinus carpio L.). In the fish infected with aeromonads, changes in the ratios of fatty acids in phospholipids are similar for either lymphocytes, liver, or whole blood. The extent to which these changes are pronounced depends on the original physiological status of the fish.     

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro

8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.     

Effect of lumpsucker (Cyclopterus lumpus L.) C-reactive protein on DNA synthesis in cultured murine and fish leucocytes

• 1.1. C-reactive protein (CRP) isolated from a marine teleost (Cyclopterus lumpus L.) an structurally resembling mammalian CRP was added to cultures of leucocytes obtained from fish peripheral blood or mouse spleens.
• 2.2. A statistically significant dose-dependent stimulation by the fish CRP, of [³H]thymidine incorporation into normal mouse spleen cells, was observed. The effect was substantially reduced in the presence of phosphorylcholine.
• 3.3. Addition of fish CRP to fish leucocytes or to cultures of nu nu mouse spleen cells did not result in significant stimulation. This suggests that although the CRP is not mitogenic for fish leucocytes it exerts a selective stimulatory effect on mouse T cells.
• 4.4. A synergistic stimulatory effect between fish CRP and PHA was abolished by phosphorylcholine and suggests that phosphorylcholine residues may contribute to the binding sites of mammalian lymphocytes.

     

Effect of Bacterial Infection on the Lipid Composition of Carp Blood Lymphocytes

The initial stage of bacterial infection is characterized by an increase in the level of total lipids and polyenoic fatty acids in membrane phospholipids of blood lymphocytes of carp (Cyprinus carpio L.). In the fish infected with aeromonads, changes in the ratios of fatty acids in phospholipids are similar for either lymphocytes, liver, or whole blood. The extent to which these changes are pronounced depends on the original physiological status of the fish.     

Porcine blood cell separation by porous cellulose acetate membranes

Leukocytes were separated from whole porcine blood using laboratory prepared polymeric asymmetric porous membranes from cellulose acetate (CA) and by applying standard blood cell separation methods: centrifugation in a Ficoll solution gradient and in sucrose solution concentration gradient. Leukocytes, obtained by different separation methods were characterised by their quantity, type, viability and growth ability. Membranes prepared by a wet phase inversion process from different cellulose acetate/acetone/water and magnesium chlorate VII systems, were characterised according to: permeability to deionised water, surface morphology and by the determination of the flux of the permeate during the whole porcine blood separation. Cellulose acetate membranes prepared from 300 μm thick cast solution (14.8 wt% of cellulose acetate, 19.9 wt% of water, 2.3 wt% of Magnesium perchlorate, and 63.0 wt% of acetone), have separation characteristics comparable with the standard separation methods; in the dead-end mode filtration, 21.3% of leukocytes from porcine whole blood are separated. The leukocyte number in peripheral blood before separation was 450,000 ml^-1; the number passed through after was 95,000±6620. The main interest of the study was to introduce the CA membrane filters for the continus technological separation of the leukocyte/lymphocytes from animal (= porcine, bovine, horse..) blood. This revised version was published online in June 2006 with corrections to the Cover Date.     

Human T lymphocyte differentiation antigens: effects of blood sample storage on Leu antibody binding

Current studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens. With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS). Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood. Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22 degrees C). With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results. When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided.     

Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.     

A review of tricaine methanesulfonate for anesthesia of fish

Tricaine methanesulfonate (TMS) is an anesthetic that is approved for provisional use in some jurisdictions such as the United States, Canada, and the United Kingdom (UK). Many hatcheries and research studies use TMS to immobilize fish for marking or transport and to suppress sensory systems during invasive procedures. Improper TMS use can decrease fish viability, distort physiological data, or result in mortalities. Because animals may be anesthetized by junior staff or students who may have little experience in fish anesthesia, training in the proper use of TMS may decrease variability in recovery, experimental results and increase fish survival. This document acts as a primer on the use of TMS for anesthetizing juvenile salmonids, with an emphasis on its use in surgical applications. Within, we briefly describe many aspects of TMS including the legal uses for TMS, and what is currently known about the proper storage and preparation of the anesthetic. We outline methods and precautions for administration and changes in fish behavior during progressively deeper anesthesia and discuss the physiological effects of TMS and its potential for compromising fish health. Despite the challenges of working with TMS, it is currently one of the few legal options available in the USA and in other countries until other anesthetics are approved and is an important tool for the intracoelomic implantation of electronic tags in fish.     

草鱼感染GCRV后血液中淋巴细胞变化及BCL10表达分析

为了研究草鱼BCL10基因在草鱼出血病中的应答机制, 文章克隆了BCL10基因, 并利用生物信息学、荧光定量和血涂片等技术对其进行了分析。生物信息学结果显示, BCL10基因开放阅读框为738 bp, 编码245个氨基酸。实时荧光定量PCR结果显示, 感染病毒后草鱼体内BCL10表达量持续上调, 在肝胰腺和中肾中第4天达到峰值, 第7天表达量开始下调。血涂片显微镜观察发现了血液中淋巴细胞在感染病毒后第1到第4天下降, 第7天时上升。肾脏的组织病理学观察也发现中肾中肾小管上皮细胞第1到第7天逐渐空泡化, 脱落坏死。以上结果表明, BCL10基因参与了草鱼应对草鱼呼肠孤病毒(GCRV)入侵的免疫应答。     

Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene-dodecanoic acid by human peripheral blood cells

The fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12-(1-pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to "gate out" nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.     

Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

A sonar fish counter

The basic principles of a new technique for counting fish moving up or down stream are given. The counter is based on the ability to detect the presence of fish in an acoustic beam directed across the river. The counter is capable of counting several fish moving up or down stream simultaneously and is designed for use in wide open channels. The results of field trials with the equipment at two different sites are discussed together with the problems encountered. The results obtained are in good agreement with other methods of counting fish but the totals obtained were insufficient to put confidence limits on the counter.