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1.
This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)‐like cells of a tiny freshwater fish Leopard danio (Brachydanio frankei). Embryonic stem (ES)‐like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz‐15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic fibroblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES‐like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage‐specific embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% significant viable ES‐like cells (P<0.05). The post‐thawed cells retained similar properties of non‐cryopreserved ES‐like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%.  相似文献   

2.
Culturing pluripotent embryonic stem cells represents a unique model system for in vitro studies of embryo cell growth and differentiation, and represents a connection between in vitro and in vivo manipulation of genes. To further develop and refine stem cell technology for marine fish, we have established cultures of embryonic stem cells isolated from turbot blastulas. The pluripotent nature of our turbot-ES-like cells was supported by their morphology and elevated levels of alkaline phosphatase enzyme activity, their ability to remain undifferentiated for a prolonged culture period, their spontaneous differentiation potential in vitro and their ability to form embryoid bodies (EB) in response to changes in the extracellular environment. In addition, we show that turbot ES like cells express Oct-4 required for the maintenance of pluripotency of ES cells. Cells from 100 blastulas (>105 cells/well) were seeded into gelatine coated 24 well cell culture clusters. The cells were polygonal in shape, with dense cytoplasm and large nuclei. The ES-like cells formed colonies within 24 h following seeding, multilayered in a pyramidal fashion, with maximum cell densities in the middle. The cells proliferated vigorously when seeding densities were high and the cells still had not attached to the gelatine-coated surface. Most of the cells became attached to the surface 48 h following seeding. Attached cells grew more slowly and 20% of the plated colonies could be kept stable for 60 days. Eventually, most of the cultures showed extensive differentiation or died. Only a few cultures (4–5%) survived prolonged culturing (>2 months). The cells were stained for alkaline phosphatase activity, a marker of pluripotency and showed intense staining. More specific, turbot ES like cells in culture expressed Oct-4, detected by immunofluorescence staining. Changing the medium conditions by adding retinoic acid and removing LIF, the proportion of embryoid bodies in our cultures increased. ES-like cells as well as fresh, intact fertilised eggs where successfully cryopreserved. ES cells from the cryopreserved eggs could be isolated and seeded into cultures, forming colonies like the cells from freshly fertilised eggs. Also cryopreserved ES-like cells could be successfully plated. The prolonged survival of these cryopreserved cells has not yet been investigated. The establishment of in vitro cultures of turbot ES-like cells represents a new experimental model for marine flatfish. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

3.
In this study, we established and characterized a cell line derived from the kidney of black carp (Mylopharyngodon piceus), which is an important freshwater aquaculture species. The cell line was designated as MPK and subcultured for more than 70 passages in DMEM medium containing 10% fetal bovine serum (FBS) at 28°C. MPK had a modal diploid chromosome number of 48. Moreover, a transient MPK transfection efficiency was up to 18% using a green fluorescent protein plasmid by a modified electroporation. In addition, the MPK cells showed susceptibility to spring viremia of carp virus (SVCV), as demonstrated by the presence of severe cytopathic effects (CPEs) and increased viral RNA. Unexpectedly, the MPK cells expressed pluripotency‐associated genes such as nanog, oct4 and vasa, indicating that these are possibly adult stem cells. Taken together, we have established a stable cell line from kidney that may potentially be utilized as an in vitro platform for genetic modifications and host–pathogen analysis in black carp.  相似文献   

4.
Piscirickettsia salmonis is an intracellular bacterium that was first isolated and identified in fish cells. Several types of cell lines have been explored for their ability to provide the bacterium with a host cell to replicate in. Tissue culture has been used for growth and cultivation for nearly two decades, until the facultative nature of P. salmonis was confirmed upon the development of blood‐ and cysteine‐based agar. Since then, research has continued to drive the creation of novel agar and broth formulations in order to improve the efficacy of cultivation of P. salmonis. Until now, the techniques and components used for growth have not been thoroughly discussed. In this review, the methods and formulations for growth of P. salmonis in tissue culture and cell‐free media will be examined.  相似文献   

5.
Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.  相似文献   

6.
陈晓武  申亚伟  赵金良  吴明林 《水产学报》2018,42(10):1626-1634
为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。  相似文献   

7.
Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.  相似文献   

8.
Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.  相似文献   

9.
Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15–20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 107 cells mL−1. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.  相似文献   

10.
  1. The use of translocations to establish new or ‘refuge’ populations for species with high conservation value is controversial but widely used in conservation management. One of the risks of this approach is that an establishing population does not adequately capture the genetic diversity of the donor gene pool. This effect, rarely examined, is tested here.
  2. In this study the genetic consequences of two conservation translocations after five generations (16 years) of the European whitefish, Coregonus lavaretus, were quantified. Both translocations were made using almost the same genetic groups and thus represent a partly replicated natural study.
  3. Analysis of 12 informative microsatellites showed that expected heterozygosity, the mean number of alleles per locus and allelic richness did not differ between donor and translocated populations. There was also no loss of heterozygosity in the translocated populations, nor deviations from Hardy–Weinberg equilibrium expectations, nor signs of linkage disequilibrium.
  4. All populations were genetically differentiated but pairwise FST values were low, indicating that the magnitude of divergence was small.
  5. There was no evidence of inbreeding but there were significant differences in private allelic richness between donor and translocated populations. Of 50 alleles found in the donor population, 16% of the rarer alleles were lost in one translocated population and 8% in the other.
  6. Allele loss without a reduction in heterozygosity strongly points to stochastic drift effects having occurred following translocation. The evidence indicates that alleles that were not detected in the donor population have arisen de novo in the translocated populations.
  7. It is concluded that conservation translocations comprising even a modest number of propagules can successfully capture a high proportion of genetic variation of the host population, and that reduced genetic variation in the translocated population may be mitigated by the emergence of new variation over short time periods.
  相似文献   

11.
The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.  相似文献   

12.
  1. Genetic information is crucial for the conservation of Dipturus oxyrinchus (Linnaeus, 1758), a threatened large skate with declining populations over most of its geographical range. The main aim of the present study was to investigate the genetic structure, connectivity and demographic history of the longnosed skate in Sardinia (western Mediterranean Sea).
  2. Patterns of population structure were assessed in 175 specimens from six sampling sites. Variation in two mitochondrial genes (cytochrome c oxidase subunit I (COI) and control region) highlighted high genetic diversity and low but significant genetic differentiation among sites, which clustered into three groups corresponding to the north‐west, north‐east and south Sardinian coasts.
  3. The observed genetic structuring could presumably depend on a combination of past geological events, contemporary restrictions to dispersal and biological characteristics of the species (e.g. site‐fidelity, no pelagic larval stage, limited dispersal of juveniles and/or adults).
  4. Demographic analyses showed signs of past population expansion, but substantial current stability of Sardinian populations. From a conservation perspective, these results are encouraging, and indicate that Sardinian populations are still large and stable, and seem not to have suffered negative side‐effects from the ever‐growing fishing pressure in the region.
  5. The occurrence of genetic structuring strongly supported the close monitoring of populations to identify any erosion of their gene pool, and high genetic variability of the Sardinian D. oxyrinchus populations could thus represent priority populations for conservation purposes, providing potential sources for recolonization in cases of local extinctions in other areas of the distribution range of the species.
  6. When the sequences from Sardinia were compared with those available from other areas, the data seem to exclude the possibility that the Atlantic and Mediterranean host totally isolated populations or even different species, as recently suggested. However, additional markers and a larger sampling sites are needed to confirm these findings.
  相似文献   

13.
Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.  相似文献   

14.
Heterosporis saurida is a microsporidian that infects lizardfish, Saurida undosquamis (Richardson, 1848), in the Arabian Sea. Spores were isolated from infected lizardfish and used to infect derived fish cell lines: common carp brain (CCB), epithelioma papulosum cyprinid (EPC), fathead minnow epithelial (FHM), rainbow trout gonad (RTG), bluegill fry (BF‐2) and chinook salmon embryo (CHSE). Non‐fish cell lines were also tested that include: insect (SF‐9), rabbit (RK‐13) and African green monkey (Vero E6). No growth of H. saurida was observed in any fish cell line, SF‐9 or Vero E6 cell lines. H. saurida spores grew only in RK‐13 cell line and were detected by immunofluorescence. Developmental stages of H. saurida were seen in RK‐13 cells by light and transmission electron microscopy, and species identification was confirmed by sequencing. This study demonstrated that H. saurida was able to proliferate in the mammalian RK‐13 cell line, which thus represents an in vitro model for conducting molecular genetics and cell–pathogen interaction studies of Heterosporis.  相似文献   

15.
为获得具有单一克隆特性的能稳定传代培养的罗非鱼巨噬细胞系,本研究从尼罗罗非鱼腹腔中分离纯化巨噬细胞,采用EB病毒(Epstein-Barr virus,EBV)感染,筛选单克隆细胞的方法建立了尼罗罗非鱼巨噬细胞系,并对其进行了EBV感染鉴定、电镜观察、端粒酶活性检测、致癌性评估、核型分析以及分子生物学鉴定。研究表明,EBV已整合到尼罗罗非鱼巨噬细胞中且稳定表达,经30代稳定传代,该细胞系仍维持较好的增殖状态;该细胞系表面不平滑,有明显的钝圆形突起和细长的伪足,表现为典型的巨噬细胞形态;端粒酶活性显著高于未经感染的巨噬细胞,而与He La细胞差异不显著,且该细胞系不具有致癌性,说明永生化细胞系构建成功。核型分析结果发现,该细胞系具有44条染色体,其核型公式为2 n=2 x=44=4 sm+17 st+1 t。PCR检测发现,该细胞系存在CD33和CD205的转录本,这些都是单核巨噬细胞的标志物,经18S r RNA检测证明该细胞系来自尼罗罗非鱼巨噬细胞。永生化尼罗罗非鱼巨噬细胞系已被成功建立,该细胞系为研究罗非鱼链球菌HSP70-肽疫苗的高保护率,以及罗非鱼的免疫防御机制提供了工具。  相似文献   

16.
A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.  相似文献   

17.
Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD) is an emerging disease that contributes to serious production losses in Macrobrachium hatcheries worldwide. Mosquito cell lines (C6/36) have been reported to support the growth of MrNV and used to observe the cytopathic effects (CPE) in infected cells. This study determined the susceptibility of C6/36 mosquito cells to the Australian isolate of MrNV in order to use fewer animals in further investigations. Different staining methods were used to observe MrNV viral activity in C6/36 cells. Typical cytopathic effects such as vacuolation and viral inclusion bodies were observed in infected C6/36 cells with H&E and Giemsa staining. With acridine orange, it was easier to detect presumptive MrNV messenger ribonucleic acid in the infected cells. Using neutral red staining to measure mitochondrial activity showed light absorption of infected cells maximized at day 4 (O.D. = 0.6) but was significantly lower (chi‐square = 41.265, df = 1, P < 0.05) than control groups (O.D. = 2) which maximized at day 12. Using trypan blue staining to count the number of cells with disrupted cell membranes, the maximum number of presumptively dead cells at day 8 (4 × 105 cells) in infected treatments was higher than the control treatment at day 10 (1.8 × 105 cells). However, TaqMan real‐time PCR did not confirm the replication of MrNV in the cells over 14 days. The mean viral copies and mean cycle times of positive samples were stable at 2.07 × 104 and 24.12, respectively. Limited evidence of viral replication was observed during four serial passages. This study determined the mortality of the C6/36 cell line to the Australian isolate of MrNV but suggests limited patent replication was occurring. Trying different cell lines or adapting the virus to the C6/36 cells may be necessary to successfully replicate Australian MrNV in cell lines.  相似文献   

18.
孙迪  丁洪昌  严兴洪 《水产学报》2018,42(4):534-543
为探讨尿囊素对印度产紫菜Pyropia chauhanii单孢子形成与放散的影响,选用能释放单孢子的野生型品系(PC-WT)与不能释放单孢子的诱变品系(PC-Y1)为实验材料,用含不同浓度尿囊素的培养液分别培养来自叶状体梢、中和基部的圆盘体。结果显示,处理后6 d,不同部位的叶状体圆盘体的单孢子放散量次序为中部梢部基部;随着尿囊素浓度的增加,PC-WT品系叶状体圆盘体的单孢子放散总量呈先升后降的趋势,其中10mmol/L是促进单孢子放散的最适浓度;但尿囊素并不能使原本不放散单孢子的PC-Y1品系释放单孢子。用含20 mmol/L尿囊素的培养液培养PC-Y1品系的叶状体圆盘体,8 d后再用酶解法将其体细胞单离出来经体外培养后发现,经尿囊素处理的单离细胞发育成丝状体的百分率为66.1%,而不含尿囊素的对照组仅为15.2%,说明尿囊素对紫菜叶状体的体细胞向生殖细胞分化具一定的促进作用。  相似文献   

19.
Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE‐214). Different stages of viral replication were observed under electron microscopy. Virus‐like particles were observed inside membrane‐bound vesicles as early as 1 h following contact of the virus with the cells. Membrane‐dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome‐like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive‐sense single‐stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by ‘fuzzy’‐coated membranes was greater in virus‐infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post‐infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.  相似文献   

20.
In vitro cultures of native fish cell lines are of great importance, both for basic research and applied science. In particular, there is strong demand for long-term growable cell lines from breeding fish, like sturgeon. Here, we describe the culture of cells from Siberian sturgeon (Acipenser baerii) head kidney. The cells have so far been cultured over a period of 12 months (24 passages). Cytochemical and immunocytochemical examination suggests that, in vitro, the cells exhibit markers that are indicative for different cell types. In particular, fat storing cells (adipocytes) were observed, and the expression of cytokeratins and glial fibrilar acidic protein (GFAP) can be concluded on the basis of immuncytochemical analysis. The observation of different morphologies additionally underlines the heterogeneity of the cell population and matches the typical behaviour of in vitro cultures of stem/progenitor cells. Different applications can be imagined.  相似文献   

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