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1.
The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical “S” shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis. 相似文献
2.
A cell line,SHK,was derived from the kidney of spotted halibut Verasper variegates.The cell line was subcultured more than 40 passages in minimum essential medium(MEM)supplemented with fetal bovine serum(FBS)and 10 ng ml-1 basic fibroblast growth factor(bFGF).Cell morphology from primary culture and subculture was observed continuously by microscopy.The SHK cell line consisted predominantly of fibroblast-like cells.The cell line was able to grow between 20℃ and 30℃ with the optimum growth at 24℃ and with a reduced growth between 12℃ and 20℃.The growth rate of the cells increased as the proportion of FBS increased from 10% to 20% at 28℃ with optimum growth at the concentration of 20%.The doubling time of the cells was determined to be 44.8 h.Chromosome analysis revealed that 52% of the SHK cells maintained a normal diploid chromosome number (2n=46).The cells were successfully transfected with green fluorescent protein(GFP)reporter plasmids and the expression of GFP gene in the cells indicated the possible utility of the cells in gene expression studies.The cells were infected by lymphosystis disease virus(LCDV)and found to be susceptible to the virus in cytopathic effect(CPE)observation.The infection was confirmed by PCR and electron microscopy experiments,which proved the existence of the viral particles in the cytoplasm of the virus-infected cells. 相似文献
3.
Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) on cartilage cells from proboscis
of skate,Raja porasa Günther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24°C. Twelve hours
after culture initiation, the cartilage cells were treated with bFGF and IGF-II at different concentration combinations. It
was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-II was enough to have obvious stimulating effect on the growth and division
of skate cartilage cells. Test of bFGF and IGF-II together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-II together
had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form
a monolayer at day 7.
This work was partially supported and funded by the China Education Committee as the “Imbursement Project for Studied Abroad
Returnees”. 相似文献
4.
Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry
causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination
technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed
after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb)
were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from
PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection
site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared
100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the
injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence
did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis
indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6–50 days post-vaccination. These results demonstrate that the
antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The
antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder
(Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could
induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for
the development and application of this promising DNA vaccine. 相似文献
5.
Effects of Basic Fibroblast Growth Factor and Insulin-like Growth Factor on Cultured Cartilage Cells from Skate Raja porasa 总被引：1，自引：0，他引：1
Effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor Ⅱ(IGF-Ⅱ) on cartilage cells from proboscis of skate, Raja porasa Glinther, were investigated in this study. The cartilage cells were cultured in 20% FBS-supplemented MEM medium at 24℃. Twelve hours after culture initiation, the cartilage cells were treated with bFGF and IGF-Ⅱ at different concentration combinations. It was found that 20 ng/ml of bFGF or 80 ng/ml of IGF-Ⅱ was enough to have obvious stimulating effect on the growth and division of skate cartilage cells. Test of bFGF and IGF-Ⅱ together, revealed that 20 ng/ml of bFGF and 80 ng/ml of IGF-Ⅱ together had the best stimulating effect on the growth and division of skate cartilage cells. The cartilage cells cultured could form a monolayer at day 7. 相似文献
6.
Fe^2＋ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe^2＋ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe^2＋. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually. 相似文献
7.
The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and
morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained
various initial concentrations of Pb2+ and Cd2+ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure,
growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb2+) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated
or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest
concentration (8 mg/L Pb2+), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2,
3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb2+), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb2+, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd2+concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC50) values (3.47 mg/L Cd2+ and 12.11 mg/L Pb2+) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd2+ than Pb2+.
Supported by the Chinese Scholarship Council 相似文献
8.
To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against
human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin
(PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like
factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes
and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation
assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2,
(rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface
of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected
with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.
This work was supported by National “973” Project G1999012003, G19999012006. 相似文献
9.
The early stage differentiation of thallus cells ofPorphyra haitanensis T. J. Chang et B. F. Zheng was studied. Protoplasts or single cells were isolated from the blades using enzyme mixture comprising
2% sea snail gut enzyme and 1% cellulase. The isolated protoplasts or single cells were incubated in the MES medium. The cell
differentiations were examined under the microscope at intervals after incubation. Four types of cell differentiation, namely,
normal, abnormal, carposporangial and spermatorangial, and rhizoidal types, were observed. Since normal cell differentiations
occur mostly in small thalli 50 mm in length and middle portions of big thalli 200 mm in length, it is essential to select
tissues from these two kinds of thalli essential for commercial production. 相似文献
10.
11.
【目的】建立可用于大规模培养的户外开放式微藻培养体系。【方法】设计一种微藻平面开放浅层培养(Flat,open and shallow,FOS,简称浮法)体系,以小球藻(Chlorella sorokiniana)为模式藻种,探讨温度、光照、pH值和培养基营养成分等因素在该培养体系中对小球藻生长的影响,开展900 L体系的户外培养试验。【结果】浮法体系主要由塑料袋和垫板组成,在该培养体系中的小球藻在20~40℃范围内均可生长,最适生长温度为30~35℃。小球藻生长有明显的照度依赖性,无光时几不生长,随着照度升高,生长速度加快,在较高的照度下表现有光饱和现象。以TAP为基础培养基,在碳源或氮源缺乏时小球藻几不生长。pH 7.5左右有利于小球藻生长。900 L体系的户外培养试验的生物量(干物质得率)为0.15 g/(L·d)。【结论】在此新型户外平面开放浅层微藻培养体系中,温度、照度、pH值及营养成分等对小球藻的生长均有不同程度的影响。该体系有成本低、操作简便、容易控制、自然资源利用率高等特点,有大面积推广潜力。 相似文献
12.
We isolated a strain of lymphocystis disease virus (LCDV) from Japanese flounder (Paralichthys olivaceus) cultured in northern China. Based on published sequences of major capsid protein (MCP) gene of LCDV-cn (GenBank: AF126405),
we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization (ISH) to detect
LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral
supernatant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA
sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus (OFIV and RSIV), Iridovirus (CzIV and WIV), Ranavirus (TFV and FV3), and Lymphocystivirus (8 LCDV). Based on the alignment, we confirmed the PCR product was from Lymphocystivirus (GenBank accession number DQ279090 (LCDV-HD)). Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen,
stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel’s
black rockfish (Sebastes schlegeli Higendorf), redwing sea robin (Lepidotrigla microptera Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method. 相似文献
13.
In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI. 相似文献
14.
以全鱼粉作为唯一蛋白源(D1),用豆粕替代10%、20%鱼粉(D2、D3),玉米蛋白粉替代10%鱼粉(D4),啤酒酵母替代10%鱼粉(D5),配制5组等氮等能饲料,每种饲料设置3个实验组,进行56 d的养殖实验。通过血液和组织涂(印)片、细胞染色和显微观察,研究人工培育的褐点石斑鱼幼鱼外周血液白细胞的分类组成,头肾、脾脏、体肾和肝脏等4种组织中各类血细胞的发生情况,以及不同蛋白源饲料对褐点石斑鱼血细胞发生的影响。结果表明:褐点石斑鱼外周血液中的白细胞由淋巴细胞(53.30%±4.66%)、血栓细胞(35.69%±3.85%)、嗜中性粒细胞(10.34%±3.14%)、单核细胞(0.28%±0.36%)、浆细胞(0.24%±0.34%)和嗜酸性粒细胞(0.15%±0.27%)组成;组织印迹片中,未成熟的红细胞、淋巴细胞和粒细胞主要在头肾印迹片中出现,未成熟的单核细胞主要在头肾和脾脏印迹片中出现,血栓细胞在肝脏印迹片中数量最多,推断褐点石斑鱼幼鱼主要的造血组织是头肾,其次是脾脏;在4种组织中均观察到浆细胞,在体肾印迹片中观察到嗜碱性粒细胞,在肝脏印迹片中观察到巨噬细胞,在头肾印迹片中还观察到巨大原红细胞。显微观察和数据统计分析的结果都表明,投喂5种蛋白源不同的配合饲料,未对褐点石斑鱼4种组织中血细胞的发生情况造成显著影响。 相似文献
15.
Tingjun Fan Xiuzhong Hu Liyan Wang Xiaofen Geng Guojian Jiang Xiuxia Yang Miaomiao Yu 《中国海洋大学学报(英文版)》2012,11(1):65-69
Turbot(Scophthalmus maximus L.) reddish body iridovirus(TRBIV) was propagated in turbot fin cells(TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study.TF cells were cultured in 10% bovine calf serum(BCS)-containing MEM medium(pH7.0) at 22℃,in which TRBIV propagated to a titer as high as 105.6 TCID50 mL-1.The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide.The inactivated vaccine caused neither cytopathogenic effect(CPE) on TF cells nor pathogenic effect on turbots.After being administered with the vaccine twice via muscle injection,the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner.The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms,respectively.The inactivated vaccine was very immunogenic,efficiently preventing tur-bot from death.It holds the potential of being applied in aquaculture. 相似文献
16.
Chinese shrimp (Fenneropenaeus chinensis) is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism
partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization (FISH)
was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene (rDNA) of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of
homologous chromosomes in F. chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate
the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F. chinensis. The successful application of FISH in F. chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes
of penaeid shrimps. 相似文献
17.
A genetic transformation model for the seaweedLaminaria japonica mainly includes the following aspects:
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1.
The method to introduce foreign genes into the kelp,L. japonica
Biolistic bombardment has been proved to be an effective method to bombard foreign DNA through cell walls into intact cells of both sporophytes and gametophytes. The expression ofcat andlacZ was detected in regenerated sporophytes, which suggests that this method could induce random integration of foreign genes.
Promoters to drive gene expression
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2.
The CaMV35S promoter was first used by us to induce the expression of GUS gene in brown algae. But results of further studies suggested that CaMV35S could be a tissue-specific promoter. Our use of SV40 promoter resulted in both transient and stable expression oflacZ andcat in sporophytes or gametophytes. No GUS or LacZ background was found in either sporophytes or gametophytes.The regeneration route of transgenic kelp
The regeneration efficiency of explants is still very low. By using female gametophytes as gene hosts and parthenogenesis as regeneration route, CAT activity and LacZ activity were detected in regenerated sporophytes of parthenogenetic kelp. li]4.|The way to select transgenic kelp
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1.
Results of sensitivity tests showed that kelp was only sensitive to chloramphenicol and hygromycin among many antibiotics. The regenerated sporophytes by parthenogenesis were more sensitive to hygromycin than to chloramphenicol. Resistant kelp was created by transforming female gametophytes with pSV40-CAT and stimulating parthenogenesis followed by selection in medium with lethal concentration of chloramphenicol.
Safety consideration of transgenic kelp
L. japonica was originally introduced from Japan. In China it is a cultured population. The possibility of its negative impact on natural populations is very low. 2) The vectors and target genes used for transformation should be restricted in order to avoid any negative impacts on human health and environment. 3) Specially devised containers (3.6 L, made of 200 μm membrane) were used to ensure that the kelp cannot escape or be eaten by marine animals. 4) To avoid the release of spores, it is very necessary to harvest the kelp at suitable age before the sporangium forms.
18.
From 2007 to 2009, large-scale blooms of green algae (the so-called “green tides”) occurred every summer in the Yellow Sea,
China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality
of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined.
This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture
medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and
decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could
be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process. 相似文献
19.
Dietary leucine requirement for juvenile large yellow croaker, Pseudosciaena crocea Richardson 1846 (initial body weight 6.0 g ± 0.1 g) was determined using dose-response method. Six isonitogenous (crude protein
43%) and isoenergetic (19 kJ g−1) practical diets containing six levels of leucine (Diets 1–6) ranging from 1.23% to 4.80% (dry matter) were made at about
0.7% increment of leucine. Equal amino acid nitrogen was maintained by replacing leucine with glutamic acid. Triplicate groups
of 60 individuals were fed to apparent satiation by hand twice daily (05:00 and 17:30). The water temperature was 26–32°C,
salinity 26–30 and dissolved oxygen approximately 7 mg L−1 during the experimental period. Final weight (FW) of large yellow croaker initially increased with increasing level of dietary
leucine but then decreased at further higher level of leucine. The highest FW was obtained in fish fed diet with 3.30% Leucine
(Diet 4). FW of fish fed the diet with 4.80% Leucine (Diet 6) was significantly lower than those fed Diet 4. However, no significant
differences were observed between the other dietary treatments. Feed efficiency (FE) and whole body composition were independent
of dietary leucine contents (P > 0.05). The results indicated that leucine was essential for growth of juvenile large yellow croaker. On the basis of FW,
the optimum dietary leucine requirement for juvenile large yellow croaker was estimated to be 2.92% of dry matter (6.79% of
dietary protein). 相似文献
20.
Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5
and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235 bp (28.0%), 307 bp (36.6%), 138 bp (16.5%)
and 158 bp (18.9%), respectively. Furthermore, 556 bp fragment of the sequence was used to discuss the phylogenetic relationship
among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp
groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus. 相似文献