BjAB细胞感染Epstein-Barr病毒后表面形态的变化

在扫描电镜下观察BjAB细胞在感染EB病毒以后表面形态的变化。未受感染的BjAB细胞以表面布满丝状伪足者最多见。感染1天后,部分细胞丝状伪足缩短变粗,另部分则丝状伪足几乎全部消失,细胞表面略现皱褶和分布着疏散的“手指样”结构。感染后3天时细胞突出的变化是表面皱褶更明显,更多见;而且开始看到表面泡状结构。这种泡状结构的细胞在感染5天时数量增多,而7天后大部分细胞已呈“泡样瘤”状。观察感染后5个月的细胞,这种表面结构仍保持不变。由于用灭活的EB病毒在相同条件下感染这种细胞,表面形态未见改变,所以这种表面形态的变化应与EB病毒感染,并设想同EB病毒基因组的存在和表达有关。     

猪生殖与呼吸综合征病毒感染Marc-145细胞的病毒形态发生和细胞超微结构观察

以猪生殖与呼吸综合征病毒四川分离株PRRSV-SC1株感染体外培养的Marc-145细胞为模型,通过透射电镜对PRRSV的病毒形态发生学和宿主细胞超微结构的动态变化规律进行研究。结果显示,病毒粒子呈球形,有囊膜,大小约45-65nm,内含直径约25-30nm的核衣壳。病毒感染细胞后以细胞内吞方式进入细胞,在胞浆内复制,装配好的病毒以出芽或细胞外分泌释放到细胞外。感染细胞超微结构变化主要表现为:细胞胞浆空泡增多,内质网扩张,线粒体增生、嵴肿胀、脱落,最后空泡化,细胞表面的微绒毛脱落,出现典型的细胞凋亡特征,并观察到凋亡小体,最后整个细胞裂解、破碎。     

BST-2抑制HIV-1病毒样颗粒释放的研究

BST-2是最近发现的可以抑制成熟HIV-1(human immunodeficiency virus,HIV)病毒颗粒从哺乳动物细胞表面释放的宿主因子,随之发现其也可以抑制多种包膜病毒的释放。本研究采用密码子优化的表达HIV-1 gag和gag-pol蛋白的质粒所形成的病毒样颗粒作为研究对象,观测BST-2对这两种病毒样颗粒(Virus-like particle,VLP)的释放抑制情况及其作用机制。结果发现,瞬时表达和稳定表达的BST-2均可以显著抑制病毒样颗粒从哺乳动物细胞释放,同时发现这两种病毒样颗粒(gag/gag-pol)的释放都可以被BST-2抑制;而且,HIV-1中Vpu蛋白可以拮抗BST-2抑制HIV病毒样颗粒释放的作用,另外,通过化学试剂和酶学方法处理,确证BST-2可以被包装进病毒样颗粒中。     

辛德毕斯病毒6K蛋白在病毒成熟释放中的作用

应用多种细胞生物学技术,对辛德毕斯病毒(Sindbis Virus,SbV)的6K蛋白在细胞中的分布和转运动态及在病毒释放过程中的作用进行了探讨,结果表明:6K蛋白不仅存在于成熟病毒粒子中,而且在粗面内质网合成后及在向细胞质膜的转运过程中均与囊膜蛋白E2形成复合物。对6K蛋白酰基化位点突变株的研究结果表明,它与E2突变株在病毒形态发生方面有惊人的相似之处,说明SbV的出芽释放过程不仅仅是靠E2蛋白C端与病毒核衣壳的相互作用,6K蛋白的酰基化修饰可能在这个过程中也起着十分重要的作用。根据6K蛋白的二级结构的预测及综合上述实验结果,对SbV出芽释放的模式提出了新的补充。     

牛病毒性腹泻病毒的成熟和释放

试验中用电镜观察了牛病毒性腹泻病毒ＯｒｅｇｏｎＣ２４Ｖ株在感染新生牛睾丸细胞中的形态发生。成熟的病毒颗粒是直径约为５０ｎｍ的球形颗粒,内含直径约为３０ｎｍ的核心。病毒在宿主细胞的胞质内复制,通过糙面内质网膜出芽成熟。病毒可以通过外排或在细胞死亡后含有病毒颗粒的空泡崩溃而释放到胞外。     

马传染性贫血病毒Gag p9蛋白功能研究进展

对病毒复制机制研究的一个重要方面是病毒的组装和从细胞表面出芽。过去的 2 0年大量研究证实反转录病毒Gag蛋白对病毒的组装和出芽起着决定性作用。Gag蛋白的多个功能域已经被证明在病毒组装的不同时期发挥作用。马传染性贫血病毒 (equineinfectiousanemiavirus,EIAV)p9是Gag蛋白C端的一个小蛋白 ,在其之上的L域是与病毒释放直接相关的蛋白功能区域 ,L域的核心基序YPDL可与特异的病毒或细胞蛋白相互作用共同介导病毒粒子的组装和出芽作用 ,核心基序YPDL对病毒的复制能力有一定的影响。就近年来对p9功能区与病毒组装和释放关系的研究进展进行综述。     

病毒的细胞进入研究进展及其应用前景

病毒感染的初期事件包括病毒与细胞表面受体的相互作用和进入细胞的过程,而病毒的宿主细胞专一性很大程度上取决于这一阶段的专一识别特征和特殊要求。人乳头状瘤病毒、人免疫缺陷病毒和单纯疱疹病毒是感染人类的几种常见病原物,该文简要综述和讨论了与人体健康关系密切的这三种重要病毒表面的蛋白组分、宿主细胞表面受体及其相互作用和病毒的细胞进入的研究进展,以及在以病毒的细胞进入过程为靶点的抗病毒药物研发中的应用前景。     

病毒利用泛素系统生存、增殖的方式

真核生物中, 泛素系统是个复杂的体系, 主要包括泛素,26S 蛋白酶体和酶系统E1、E2 、E3。泛素- 蛋白酶体通路是细胞内非溶酶体蛋白降解的主要系统, 在许多细胞功能中发挥重要作用。最近研究发现, 许多病毒利用泛素系统为其自身服务, 这涉及病毒生活史的各个阶段并干扰宿主抗病毒反应的多种方式, 如下调细胞表面免疫分子而实现免疫逃避、调控病毒的基因转录、抑制细胞凋亡、促使病毒出芽和释放等。深入了
解病毒利用泛素系统的机制, 将为研究病毒感染机制提供新的视角, 并为药物研发提供新的靶标。     

带有L6565小鼠白血病病毒的细胞系的建立

用L6565小鼠的胸腺、脾脏、肝脏和肾脏组织,以及外周血的无细胞提取液,分别感染NIH3T3细胞,经逆转录酶活性测定,挑选出两株感染了L6565白血病病毒的细胞,并证实L6565白血病病毒感染小鼠后主要分布于血液和淋巴系统。电镜下可观察到细胞内含有A型和C型病毒颗粒,细胞的倍增时间分别为18和16小时。细胞的XC合胞试验为阳性,在光镜下未观察到细胞的形态发生转化。收集细胞内和释放到细胞培养液中的病毒并注入新生小鼠皮下,两个月左右做血象和组织病理检查,存活小鼠全部发生了淋巴细胞型白血病。     

包膜病毒样颗粒疫苗研究进展

病毒样颗粒(VLPs)是指由病毒一个或几个结构蛋白自行组装成不含病毒基因组且不能复制、不具有感染能力的病毒样蛋白颗粒,形态结构上类似完整病毒,具有与完整病毒相似的免疫原性。VLPs可以分为两大类:无包膜VLPs和包膜VLPs,包膜VLPs被源于宿主细胞的脂质包膜包裹,包膜表面含有保护性抗原纤突。主要就包膜病毒样颗粒疫苗的结构、重组表达及免疫原性等方面的研究进展进行了综述。     

Establishment of a novel ovine kidney cell line for isolation and propagation of viruses infecting domestic cloven-hoofed animal species

A sheep kidney-derived cell line, FLK-N3, was successfully established after serial (>100) passages. Persistent infection of this cell line with viruses and mycoplasma was not detected. The cells grew well and showed susceptibility to a wide variety of viruses derived from ovine, bovine, and porcine species, including orf virus, maedi visna virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine viral diarrhea viruses 1 and 2, bovine coronavirus, bovine respiratory syncytial virus, bovine enterovirus, suid herpesvirus 1, and porcine enterovirus. These results suggest that the FLK-N3 cell line could be useful for isolation and propagation of viruses that affect cloven-hoofed animals.     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

The effects of various mammalian sera on attachment efficiency and thymidine incorporation in primary cultures of mouse mammary epithelial cells

Mammary epithelial cells from 16- to 17-day pregnant BALB/c mice were cultured in various mammalian sera to determine the kind of serum which stimulates optimal attachment efficiency and thymidine incorporation. Of those sera tested, horse, bovine, lamb, goat and fetal bovine provided the highest attachment efficiency, whereas rat, mouse and human gave the lowest. Rabbit serum stimulated the highest thymidine incorporation into TCA-insoluble material with goat and rat providing the lowest. These results suggest that sera which provide the highest attachment efficiency for primary cultures are not the best stimulants of DNA synthesis and show that an inverse relationship exists between cell attachment and thymidine incorporation for any given type of mammalian serum.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Erythrocyte antigens in Norwegian goats: serological and genetic studies.

Goat alloantisera and bovine blood typing reagents were used to characterize eight erythrocyte antigen specificities in Norwegian goats by cluster analysis, absorption and family studies. Most of the goat sera were produced by injecting dams once or twice with blood cells or blood from their own kids. The characterized specificities were designated N1-N8. The two specificities N5 and N8 were recognized both by goat alloantisera and by reagents against the bovine factors E'1 and E'2 (N5) and I (N8), which are allelic factors in the bovine B-system. In goat families, the two specificities also behaved as alleles. Consequently, the locus or gene system coding for these specificities was called the B-system of goats. The six other erythrocyte antigens were provisionally assigned to six separate loci. In addition, a bovine anti-sheep R factor reagent reacted with cells from 3.3% of the goats tested, whereas a monoclonal antibody against the Forssman antigen reacted with all the goats tested.     

Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide.

An artificial antigen was prepared from 4-O-beta-I-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

A dose-dependent function of follicular fluid on the proliferation and differentiation of umbilical cord mesenchymal stem cells (MSCs) of goat

Umbilical cord (UC) has been suggested as a new source of mesenchymal stem cells (MSCs). In this report, we isolated MSCs from the fetal UC of goat and investigated their multipotency of differentiation into germ cells in vitro, in the presence of 0-20?% bovine follicular fluid (FF). The phenotypes, capacity of proliferation and expression of MSC markers were served as the indexes of multipotency of the isolated UC-MSCs, those were ascertained by growth curves, RT-PCR and immunofluorescent staining, respectively. Our results showed that the UC-MSCs shared a similar immunophenotype to those cells reported in mouse and human bone marrow MSCs, as well as some characteristics seen in embryonic stem cells (ESCs). In addition, our data also demonstrated that a dose-dependent function of FF on the states of differentiation of goat UC-MSCs. From 2 to 20?% of the FF can promote the proliferation of goat UC-MSC, especially the 5?% concentration of follicular fluid promote proliferation was significantly higher than 2?%. In contrast, higher concentration of follicular fluid (>10?%) induced goat UC-MSCs differentiation into oocyte-like cells. These findings provide an efficient model to study the mechanism on cell proliferation and germ cell differentiation in livestock using FF.     

The use of flow cytometry and fluorescein-labeled antibodies to measure specific milk proteins in bovine mammary epithelial cells

Summary A flow cytometric technique was developed to measure the relative concentration of whey protein and β-casein in individual fixed and permeabilized bovine mammary epithelial cells. Primary bovine mammary epithelial cells were compared to mammary cells isolated from explants after a 24-h incubation and a bovine mammary epithelial transfected cell line (MAC-T). Cells were incubated with rabbit anti-bovine whey protein (α-lactalbumin + β-lactoglobulin) or β-casein primary antibodies followed by a fluorescein-labeled goat anti-rabbit IgG second antibody. The number and intensity of fluorescing cells were measured using an EPICS Profile Flow Cytometer. Primary and explant cells contained 3.3 and 2.8 times more whey protein than MAC-T cells. Explant epithelial cells contained 2.9 and 5.1 times more β-casein than primary or MAC-T cells. The higher concentrations of specific proteins within the cells was attributed to either greater synthesis or reduced secretion. These data show that flow cytometry is capable of detecting differences in milk protein concentration in different mammary epithelial cell types.