不同仔猪血清浓度对金华猪耳缘组织成纤维细胞体外培养的影响

目的探讨不同仔猪血清浓度对金华猪耳缘组织成纤维细胞体外培养的影响,初步确定体外培养时仔猪血清浓度。方法采用组织块贴壁培养法在含5%、10%、15%浓度的仔猪血清培养基中培养,观察其细胞的形态,并采用MTT法进行细胞增殖试验。结果含10%、15%仔猪血清培养液相对于含5%仔猪血清培养液的条件下,细胞从组织块迁移出来时间短,传代的成纤维细胞,增殖迅速,细胞活力强,MTT值明显增高(P 0. 05),10%血清与15%血清比较,差异无显著意义(P 0. 05)。结论说明在10%仔猪血清浓度的条件下,采用组织块贴壁培养法进行体外培养,传代细胞活力强、增殖迅速。     

鳜下丘脑神经元细胞培养

通过建立可行,高效的下丘脑神经元细胞技术,为研究鳜鱼的摄食机理与能量代谢奠定基础。分离鳜鱼下丘脑组织,Ⅰ型胶原酶将组织消化成单细胞悬液,用完全培养液进行培养,在培养的第3d、4d、5d、6d观察细胞的形态,结果显示培养第3d时细胞贴壁量少,胞体小并且呈单个分布,在培养第4d细胞的数量显著增多,且具有典型的神经元的形态,胞体饱满,第5d形神经元胞体的融合度进一步增大,突起增长增粗,许多分支互相交错连接而形成密集的神经纤维网络,第6d细胞活性减弱,开始走向凋亡;CCK-8法检测细胞活性,结果显示在培养第5d细胞活性最高;并采用两种方法对神经元细胞进行鉴定,结果显示RT-PCR检测发现培养的下丘脑细胞可以表达神经元特异基因noggin,经免疫荧光鉴定下丘脑神经元细胞纯度为95.9%。这些结果表明通过此培养方法能够获得纯度较高的鳜鱼下丘脑神经元细胞,为进一步在细胞水平研究鳜鱼的摄食机理与能量代谢相关机理奠定基础。     

不同供核细胞、处理方法及激活时间对猪核移植重构胚发育的影响

在不同的外界因素下,使用胞质内体细胞核注射的方法,为进一步提高猪体细胞核移植的效率来寻找新途径。利用屠宰场卵巢采集的卵母细胞经过去核,注核及激活等操作后,研究猪颗粒细胞和胎儿成纤维细胞,血清饥饿处理法及不同的激活时间等因素对猪核移植重构胚体外发育的影响.结果表明,猪颗粒细胞和胎儿成纤维细胞用作核供体与猪卵母细胞构建重构胚,在卵裂率上无显著性差异。作为供核细胞的猪成纤维细胞,经过血清饥饿与不经过血清饥饿培养,核移植重构胚的卵裂率无显著性差异。在进行核注射后,使供核细胞与胞质受体作用适当的时间(1～6h)在进行激活,有利于重构胚的发育。提示颗粒细胞和猪胎儿成纤维细胞用作核供体与猪卵母细胞构建重构胚以及作为供核细胞的猪成纤维细胞,经过血清饥饿与不经过血清饥饿培养处理后,这二者对猪核移植重构胚发育的均无明显影响。但是使供核细胞与胞质受体作经过适当的时间(1～6h)的激活,却有利于重构胚的发育。     

中国对虾幼体中肠腺的结构和细胞化学研究

用组织学、细胞学以及细胞化学等手段对中国对虾中肠腺的结构发生和功能进行研究。结果表明，中肠腺始于Ｎ４－５期，为中肠前端先后向两侧突出的２对盲囊，第１对中肠腺盲惠至Ｍ３期退化消失，成体中肠腺由第２对中肠腺盲囊发育而成。中肠腺小管上 Ｚ２期细胞已有了分化，由４类细胞组成，即胚细胞（Ｅ细胞）、纤维细胞（Ｆ细胞）、泡状细胞（Ｂ细胞）和吸收细胞（Ｒ细胞）。电镜下，腺上皮细胞在发育的各个时期基本一致；Ｅ细胞的     

鸡干扰素生产条件的比较研究〈一〉

本试验用鸡白细胞。鸡脾细胞、鸡胚成纤维细胞以Ge－132、NDV－F、PHA、PolyI:C为诱生剂，诱生外源性IFN，将鸡胚成纤维细胞—水泡性口炎病毒作鸡IFN的检测系统，对不同种细胞、不同的细胞浓度、不同的IFN诱生剂及培养方法等诸因素对IFN产生的影响进行了比较：研究了Ge-1322、NDV－F、PHA、PolyI：C在鸡胚成纤维细胞上的最佳诱生剂量；同时也进行了不同诱生剂在不同细胞上产生的IFN的理化性状研究，结果表明：（1）在Ge－132、NDV－F、PHA、PolyI：C四种诱生剂中，诱生能力差异极显著（P＜0.01），且以Ge－132诱生IFN的能力为最强，其他依次为NDV－F、PolyI：CPHA（2）鸡脾细胞和鸡白细胞产生的IFN效价高于鸡胚成纤维细胞；（3）鸡脾细胞和鸡白细胞悬浮培养比静置培养产生的IFN效价高近1倍；（4）雏鸡脾细胞比成鸡脾细胞产生的IFN效价高；（5）未免疫鸡鸡胚成纤维细胞比免疫鸡鸡胚成纤维细胞产生IFN效价高；（6）鸡胚成纤维细胞在IFN诱生剂的作用下，4h即可产生IFN，20－24H达到最高产量；（7）IFN诱生剂最佳诱生剂量；鸡NDV－F为128HAU／ml;Ge－132为70ug／mlPolyI：C为50ug／ml;PHA为40ug／ml     

驼背鲈肝脏结构的光镜和透射电镜观察

采用HE、Mallory氏三色法、Heidenhain氏Azan染色,PAS反应,组织块银浸染方法,对组织切片染色处理,应用光学显微镜和透射电镜对驼背鲈肝脏的显微和超微结构进行了观察,结果表明驼背鲈肝组织中肝细胞界限不明显,不存在双核现象,有暗细胞和亮细胞之分,胞质中细胞器丰富,有大量的线粒体、内质网和溶酶体,高尔基体很难观察到,仅在胆小管和狄氏间隙的附近发现。此外,在肝血窦的附近观察到成纤维细胞、枯否氏细胞和贮脂细胞。肝组织中胆管数量众多包括肝叶中央胆管、支胆管、小叶间胆管和小叶内胆管,并且在中央静脉附近偶见辐射状排列的肝细胞索。     

克氏原螯虾不同发育时期5-HT与DA在卵巢和肝胰腺中的分布

为了研究5-HT与DA阳性物质在克氏原螯虾(Procambrus clarkii)不同发育时期的卵巢细胞和肝胰腺细胞中的分布特征,运用免疫组化法对卵巢发育4个不同时期的卵巢与肝胰腺进行观察。结果表明,各期卵巢卵细胞和肝胰腺细胞中均存在5-HT与DA阳性物质。Ⅰ期卵巢5-HT阳性物质分布于卵母细胞胞核,胞质未出现阳性;Ⅱ期～Ⅳ期5-HT阳性物质主要集中在卵母细胞胞质以及胞核。在发育各期的肝胰腺中5-HT的免疫物质均呈阳性。Ⅰ～Ⅳ期卵巢中,DA阳性物质主要分布于卵母细胞的胞核以及肝胰腺细胞,其强度呈递减趋势。结果提示5-HT具有促进卵巢发育作用,而DA则抑制卵巢发育。     

曼氏无针乌贼的卵子发生及卵巢发育

采用组织学方法对人工养殖曼氏无针乌贼(Sepiella maindroni)卵子发生、卵巢发育周期进行组织学、细胞学观察。根据细胞大小、胞核形态及卵母细胞与滤泡细胞的关系,将曼氏无针乌贼卵子发生划分为卵原细胞期、卵母细胞期、成熟期和退化吸收期4个阶段,并阐述了各期卵母细胞的组织学特征。曼氏无针乌贼卵子发生过程具有种的特异性,大约孵化出膜12 d的乌贼可见有卵原细胞,此后进入增殖期。卵母细胞属于典型的滤泡型,发育过程中同时存在同步性与异同步性;卵母细胞无初级卵膜,只有次级卵膜和三级卵膜,次级卵膜由滤泡细胞分泌,三级卵膜由输卵管腺、缠卵腺以及墨囊分泌物共同构成。通过对卵巢的外观形态和组织学观察,将曼氏无针乌贼卵巢发育划分为6个时期。     

鲁西黄牛及其杂交牛育肥试验报告

育肥牛日增重由大到小依次为西鲁、夏鲁、利鲁杂交牛和鲁西黄牛;牛的不同体重阶段的日增重有明显差别,鲁西黄牛在300kg之前和370～480kg阶段,其杂交牛在350kg之前和400～550kg阶段增重较快;北方在低温季节,若不加强保温,对育肥牛的生长不利。     

泥蚶血细胞耐饥饿及抗菌力特性的研究

显微镜下观察泥蚶血细胞抗菌能力以及在不同饥饿条件下细胞形态结构的变化。结果表明:经饥饿处理后,泥蚶血细胞随着时间的迁移,出现细胞形态不规则、胞质色泽变浅、胞质颗粒物减少、胞膜边缘光滑度减小等现象。血细胞对大肠杆菌、奥斯陆莫拉氏菌、脲放线杆菌均具较高的噬菌率,噬菌实验过程中血细胞形态结构以及细胞组分不断产生变化。细胞抗菌活性检测结果表明:血细胞对三者都具有一定的抗菌活性,活性依次为:大肠杆菌>奥斯陆莫拉氏菌>脲放线杆菌。     

河蚌外套膜的组织培养

本文报道了褶纹冠蚌和背角无齿蚌外套膜的组织培养结果。试验表明，河蚌外套膜的上皮细胞和结缔组织的成纤维细胞能在离体培养条件下生长繁殖。在试验中还能观察到所培养的组织块上皮细胞，能分泌出茶褐色的有机珍珠质，使组织块逐渐变成茶褐色。     

福寿螺足组织和外套膜组织细胞培养的初步研究

采用改良的M199培养基,对福寿螺(Ampullaria gigas Spix)足组织和外套膜组织细胞进行了体外培养研究。结果显示:接种6 h后,细胞开始从组织块中迁出,培养至第3天,迁出细胞在组织块周围形成生长晕,第21~28天,细胞覆盖大部分培养瓶底壁。足组织和外套膜组织细胞分别被传至8代和9代。足组织和外套膜组织细胞在原代培养物中主要有两类,一为上皮样小细胞,形态为圆形、椭圆形或多边形,直径7~15μm;另一类为上皮样中型和大型细胞,形态椭圆形或多边形,直径15~30μm。上皮样小细胞数量多,增殖速度较快,在原代和传代培养的中后期皆可形成细胞单层。外套膜组织细胞的生长和增殖能力高于足组织细胞,其贴壁性也好于足组织细胞,Hoechst荧光染色显示外套膜组织细胞的细胞凋亡指数明显低于足组织细胞。结果表明,贝类外套膜组织有潜力成为建立连续细胞系的组织来源。     

Development of a black sea bream fibroblast cell line and its potential use as an <Emphasis Type="Italic">in vitro</Emphasis> model for stress protein studies

A fibroblast cell line (BSF) derived from a caudal fin explant of black sea bream (Mylio macrocephalus) was developed. The optimum fetal bovine serum (FBS) concentration for fibroblast cell line growth was found to be 15–20% v/v FBS and the optimum temperature range for growth was found to be 26–30 °C. The fibroblast cells displayed a diverse distribution in chromosome number with two modal chromosome numbers of 48 and 54. Upon acute heat shock (+8 °C) the cells displayed a 4.1 fold increase in hsp70 and this elevation was not prolonged as hsp70 returned to near basal levels following a 6 h recovery period. The effect of the hsp70 inducer L-azetidine- 2-carboxylic acid was tested and it was found that at a concentration of 10 mM this inducer caused a 2.3 fold increase in hsp70 levels. The sensitivity of the fibroblast cell line to heavy metal exposure was tested by treatment with Cu2+ and it was found that hsp70 was significantly elevated in the presence of micromolar concentrations of Cu2+. The data from this study demonstrates that the established black sea bream fibroblast cell line could serve as a useful in vitro model for stress protein studies.     

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).     

脊椎动物源细胞因子对合浦珠母贝外套膜细胞培养及其矿化功能的影响

筛选出能促进合浦珠母贝外套膜细胞培养及矿化功能的细胞因子,以作为优化本物种细胞培养基的参考,挑选三龄合浦珠母贝,获取其外套膜进行原代细胞培养。向各实验组细胞培养基中分别添加表皮生长因子(EGF)、内皮细胞生长添加剂(ECGS)、胰岛素样生长因子-1(IGF-1)和碱性成纤维细胞生长因子(bFGF),通过比较细胞活性、贴壁能力、迁移能力和4种基质蛋白基因(pif80、n16、msi7及accbp)表达水平的变化,来评判这些因子对细胞培养及细胞矿化功能的影响。结果显示:(1)EGF能显著提高原代培养细胞的活性、贴壁能力和迁移能力,并促进pif80、n16和accbp基因的表达;(2)ECGS能增强细胞的贴壁能力和迁移能力,并大幅提高pif80、n16和msi7基因的表达水平;(3)IGF-1能显著增强细胞活性、贴壁能力、迁移能力和msi7的基因表达水平,但对pif80、n16和accbp基因的表达有一定抑制作用;(4)碱性成纤维细胞生长因子能增强细胞的活性及贴壁能力,并显著提升pif80、n16和accbp基因的表达水平。研究表明,脊椎动物源细胞因子具有延长细胞培养时间、增强细胞活性及促进矿化相关基因表达的作用,能够用于优化合浦珠母贝外套膜细胞的培养基。     

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.     

Relationships between intracellular bacteria and the bivalve killer dinoflagellate Heterocapsa circularisquama (Dinophyceae)

ABSTRACT: Relationships between the growths of five different clonal strains of Heterocapsa circularisquama and intracellular bacteria were investigated using culture experiments. Although each H. circularisquama strain culture was established from one cell by repeated and careful washings with micropipettes, intracellular and extracellular bacteria in each strain culture were still observed under a epifluorescence microscope using the DAPI (4',6-diamidino-2-phenylindole) staining technique. The extracellular bacteria are derived, presumably, from the inside of algal cells after the death and collapse of algal cells in culture. Using an electron microscope, bacteria were constantly observed in the cytoplasm and food vacuoles of H. circularisquama cells. The growth of five algal strains containing bacteria was compared with that of a bacteria-free strain using culture experiments under combined conditions of five different light intensities and five different strengths of culture medium. Bacteria showed no significant effect on the growth or survival of the algal cells. During the algal exponential growth phase, the intracellular bacterial cell numbers per algal cell decreased, whereas the total bacterial cell density in each algal culture increased. Final cell yield (total number) of the intracellular and extracellular bacteria varied considerably according to the algal strains. These results suggest that the intracellular bacteria of H. circularisquama grow or survive depending on the host alga, and that the alga can grow independently.     

三角帆蚌外套膜细胞体外培养优化及体内植入培养对细胞生长的影响

以DMEM为基础培养基, 通过优化改良培养基及缓冲液的配方, 对三角帆蚌(Hyriopsis cumingii Lea)外套膜进行细胞培养, 并且以显微观测、RNA/DNA的比值作为该细胞增殖的评价指标, 分别对体外培养细胞的迁出时间、速度、数量以及增殖活力进行测定。分别取体外培养第24、108、120 小时的细胞进行Hoechst DNA荧光标记, 然后将3组标记细胞植入三角帆蚌外套膜中在蚌体内培养, 在植入后第24、72、120、168、216 小时运用RNA/DNA指标检测活体培养的细胞增殖活力。结果表明, 经过优化后的缓冲液和培养基更有利于细胞从外套膜组织中迁出, 迁出速度和细胞数量显著增加(P<0.05), 且细胞的活力随着培养时间的延长而逐渐增大, 培养至108 h时, 细胞活力达到最大, RNA/DNA比值为 24.53, 在108 h后显著下降(P<0.05), 显微观测的细胞生长状况与RNA/ DNA指标测定相吻合。对体外培养的细胞植入蚌体外套膜中再进行培养时发现, 对体外培养活力较好的细胞, 活体内环境可增大细胞的活力, RNA/DNA比值最高达到25.45, 但在活体培养168 h 后活力显著下降(P<0.05); 而对体外培养活力较差的细胞, 活体内环境对细胞活力影响不显著(P>0.05)。本研究旨在为三角帆蚌外套膜细胞增殖的深入研究及建株提供基础性资料。

     

Growth characteristics of the intracellular pathogen,Piscirickettsia salmonis,in tissue culture and cell‐free media

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.