中华鳖5种组织细胞的离体培养实验

采用组织块移植培养技术,对来源于中华鳖(Trionyx sinesis)肝脏、脾脏、肾脏、心脏、皮肤及裙边组织细胞进行了原代培养,细胞均可从组织块中迁出并生长成单层细胞。对原代培养的单层细胞用胰酶-EDTA消化后传代培养,初步建立了可连续传代的中华鳖肝脏、脾脏、肾脏、心脏及皮肤组织细胞系;还对细胞的培养条件、冷冻保藏与复苏、染色体数目等进行了研究,初步确定中华鳖细胞培养的条件为32℃,MEME或R1640培养基,血清浓度为20%(V/V)。二甲基亚砜(DMSO)冻存保藏后,细胞复苏生长良好,中华鳖细胞染色体数目为2n=66。     

中华鳖５种组织细胞的离体培养实验

采用组织块移植培养技术,对来源于中华鳖(Trionyx sinesis)肝脏、脾脏、肾脏、心脏、皮肤及裙边组织细胞进行了原代培养,细胞均可从组织块中迁出并生长成单层细胞.对原代培养的单层细胞用胰酶-EDTA消化后传代培养,初步建立了可连续传代的中华鳖肝脏、脾脏、肾脏、心脏及皮肤组织细胞系;还对细胞的培养条件、冷冻保藏与复苏、染色体数目等进行了研究,初步确定中华鳖细胞培养的条件为32℃,MEME或R1640培养基,血清浓度为20%(V/V).二甲基亚砜(DMSO)冻存保藏后,细胞复苏生长良好,中华鳖细胞染色体数目为2n=66.     

青海湖裸鲤资源恢复至3.9万吨

<正>青海湖裸鲤(湟鱼)是青海省珍贵保护动物,它在青海湖"鱼鸟共生"系统中处于核心地位。近年来,青海省加大青海湖裸鲤增殖放流,对非法捕捞贩卖湟鱼违法行为严厉打击,使得青海湖裸鲤资源得到保护。截至2013年底,青海湖裸鲤资源蕴藏量达到3.9万吨,数量为1.66亿尾。值得一提的是,人工增殖放流对青海湖裸鲤资源恢复的贡献率达到22.3%。青海湖裸鲤是青海湖中唯一的水生经济动物,为青海省二级保护动物。1979年在国务院《水产资     

黄芪对鲤头肾中巨噬细胞的增殖和一氧化氮产量的影响：离体研究

用密度梯度离心分离鲤头肾中的巨噬细胞和嗜中性粒细胞。用RPMI细胞培养液对分离的细胞进行原代培养,在细胞培养液中加入不同浓度的黄芪水提取液来研究黄芪对鲤头肾中免疫细胞非特异性免疫应答的影响。黄芪水提取液单独使用时能刺激鲤头肾中巨噬细胞的增殖,但不能刺激巨噬细胞的氮暴发活性。用黄芪和脂多糖(LPS)混合刺激巨噬细胞和中性粒细胞时,黄芪水提取液能显著提高脂多糖刺激所产生的一氧化氮的产量。研究结果表明,黄芪不仅能刺激巨噬细胞数量的增加,而且能协同脂多糖增强头肾中免疫细胞的功能,从而对机体的非特异性免疫起调节作用。     

中国蛤蜊的组织培养

用1640培养基，对中国蛤蜊(Mactra chinensis)的鳃和外套膜组织进行了原代培养。实验结果表明．在温度26-27℃、pH7．4、培养基中不添加小牛血清的条件下，中国蛤蜊的组织能正常生长。鳃组织培养24小时后即有游离细胞从组织块迁移出来，最长培养时间可持续至第12天；外套膜组织培养48小时后即出现游离细胞．最长培养时间可持续至第14天。     

敌百虫对青海湖裸鲤组织蛋白质作用的初步研究

<正>青海湖裸鲤,属鲤形目、鲤科、裂腹鱼亚科,青海湖裸鲤属。青海湖裸鲤是青海湖中唯一的经济鱼类,处于青海湖整个生态系统的核心地位。受人类活动的影响,青海湖流域水土流失加剧以及旅游业和农、牧、渔业等的发展,使青海湖裸鲤的生存环境受到了严重的影响与改变。因此,人工养殖青海湖裸鲤成了一种趋势。敌百虫是一种高效低毒类杀虫剂,广泛用于防治鱼体外寄生的吸虫、肠内寄生的蠕虫和甲壳动物引起的鱼病,其常用剂量为0.2~0.5毫克/升。但它在杀死敌害生物、治疗鱼类疾病的同时,对养殖鱼类也有一定的毒害作用。目前对敌百虫的毒性研究多集中于探讨安全浓度范围方面,杨杰全等发现青海湖裸鲤对敌百虫的安全浓度为2.58毫克/升,     

青海湖裸鲤对三种药物的敏感性初探

在水温为16.2～17.4℃的静水试验条件下,用水生生物毒性试验方法,以青海湖裸鲤幼鱼为试验对象,用敌百虫、硫酸铜和高锰酸钾对其进行急性毒性试验.并求出半数致死浓度和48 h安全浓度.48 h安全浓度:硫酸铜0.112 mg/L,高锰酸钾0.22 mg/L、敌百虫2.58 mg/L.结果表明青海湖裸鲤对3种药物的敏感性...     

青海湖裸鲤人工繁殖及鱼苗培育技术的研究

为保证青海湖裸鲤 (Ｇｙｍｎｏｃｙｐｉｓｐｒｚｅｗａｌｓｋｉｉｐｒｚｅｗａｌｓｋｉｉ)原种种质、增殖放流 ,增加青海湖裸鲤资源量 ,我们于 1996～ 1998年对裸鲤的人工繁殖和苗种培育技术作了详尽的研究。经过三年试验 ,形成了《青海湖裸鲤原种生产操作规程》 (草案 ) ,掌握了苗种繁殖、孵化和培育技术 ,授精率、孵化率、出苗率平均达到 80 %以上。并对青海湖裸鲤胚胎发育期和出膜后的仔鱼期做了详尽的观察。现将试验情况报道如下 :1　基本情况1996～ 1998三年间 ,在青海省鱼类原种良种场进行了裸鲤人工繁殖试验。试验材料采自布哈河、沙…     

斑点叉尾(鱼回)肾脏组织细胞系的建立及其生物学特性

采用组织块移植培养技术,对来源于斑点叉尾(鱼回)(Icttalurus punctatus Rafinesque)肾脏组织的细胞进行原代培养,建立了斑点叉尾(鱼回)肾脏组织细胞系,定名为CCK,目前已稳定传代培养70多次.斑点叉尾(鱼回)肾脏组织细胞为成纤维样细胞,最佳培养基为M199,最适培养温度范围为28～32℃,培养基血清体积分数为10%.在此条件下,CCK细胞的倍增时间为38.9～41.0 h.斑点叉尾(鱼回)肾脏细胞的集落形成效率为(74.16±3.54)%,第9代传代细胞的染色体数目为正常二倍体2n=58,第33代传代细胞的染色体众数为60.液氮冷冻保存6个月后复苏的细胞经台盼兰染色检验,(86.69±1.04)%的细胞仍保持活性,细胞复苏后培养生长旺盛.通过对第17代离体培养细胞的28S rRNA基因进行PCR扩增及序列分析与比对,表明该细胞系来源于斑点叉尾(鱼回).[中国水产科学,2009,16(1):75-81]     

急性低氧胁迫对青海湖裸鲤血液指标和肝脏氧化应激及糖代谢的影响

为探究青海湖裸鲤(Gymnocypris przewalskii)血液和肝脏组织对急性低氧胁迫的生理响应,将体质量(25.3±3.4)g的1龄青海湖裸鲤放在透明密封的(长33 cm×宽22 cm×高18 cm)水槽中,每槽22尾。急性低氧处理前,水槽持续充氧,水体溶解氧浓度为(8.1±0.2)mg·L^-1(对照组)。急性低氧胁迫时,终止充氧,用透明塑料薄膜覆盖水槽上方,水槽内水体溶解氧浓度快速下降(试验组)。当半数青海湖裸鲤出现如行动迟缓、反应迟钝、身体失衡等缺氧症状时[约(43±2)min,溶解氧约为(0.46±0.03)mg·L^-1],测定水体溶解氧浓度,同时取样测定青海湖裸鲤血液生理生化指标和肝脏抗氧化及糖代谢相关指标的变化。结果表明,在急性低氧胁迫后,青海湖裸鲤血液生理生化指标均呈现出不同程度的变化趋势。其中Na⁺、K⁺、Mg²⁺、Cl^-和尿素氮(BUN)浓度与胁迫前相比无显著差异(P>0.05),Ca²⁺浓度显著高于胁...     

鲫肠道上皮细胞原代培养方法的研究

探讨了健康鲫(Carassius auratus)幼鱼肠道上皮细胞的原代培养方法。结果显示,用含有抗生素、胶原蛋白酶Ⅰ、EDTA和胶原蛋白酶Ⅳ组成的D-Hanks液消化无菌分离的鲫肠道可以获得大量细胞团及绒毛隐窝;用含有抗生素、胎牛血清(FBS)、表皮生长因子(EGF)、胰岛素的DMEM培养液进行培养,并根据肠道上皮细胞与成纤维细胞贴壁时间的差异进一步纯化,连续培养12d后可以得到纯化的肠道上皮细胞。     

黄颡鱼吻端细胞培养研究

采用M199培养基对黄颡鱼(Pelteobagrus fulvidraco)吻端组织进行体外培养研究,继代培养细胞传至第8代。实验结果显示:吻端组织接种培养3~5 h,细胞迁移伸展,6 d后生长细胞集落汇合,每4~6 d递次进行继代培养,培养生长的细胞呈纤维样。比较继代细胞在不同温度(15、27、37℃)和血清浓度(0、10%、20%)条件下的生长,27℃、10%血清的培养条件最适宜吻端细胞的增殖。通过染色体组型分析,证明离体吻端细胞为正常的二倍体细胞。研究结果表明,黄颡鱼离体吻端细胞对温度等培养条件有很强的适应性,培养细胞增殖迅速,具有建立连续细胞系的潜力。     

Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV)

A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.     

Establishment and characterization of two new cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel)

Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets ofmicrosatellite markers of flounder. Optimal growth temperature was 20 degrees C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses.     

Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)

Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz‐15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8–10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24–32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (?196°C).     

Establishment of a novel fin cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its viral susceptibility

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.     

Establishment,characterization, virus susceptibility and transfection of cell lines from cobia,Rachycentron canadum (L.), brain and fin

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.     

Comparative assessment of growth of Trypanosoma danilewskyi (Laveran & Mesnil) in medium containing fish or mammalian serum

The objective of this study was to determine the growth of Trypanosoma danilewskyi (Laveran & Mesnil, 1904) from goldfish, Carassius auratus (L.), in medium containing no serum, or in medium supplemented with either 10% fish serum (goldfish, carp, or tin foil barb) or 10% mammalian serum (horse or foetal bovine). After 10 days, the number of trypanosomes in flasks containing tin foil barb serum increased by nearly 700% over the initial inoculum. Similarly, a substantial increase in the number of parasites was seen after 10 days in the cultures containing carp and goldfish serum. After 6 weeks, there was more than a 15-fold increase of T. danilewskyi in cultures containing goldfish serum. Medium containing no serum or mammalian serum failed to support the growth of parasites. Therefore, serum-related factors within fish blood are required for the propagation of T. danilewskyi isolated from infected goldfish. Since T. danilewskyi can be propagated in vivo and in vitro in the presence of homologous proteins, cultured and wild-type forms can be compared to determine if cultured parasites can be used as analogues of natural life-cycle stages.