鸡胚及禽类细胞系在生物医药领域的应用及研究进展

鸡胚因发育过程清楚，长久以来作为基础和应用科学研究领域重要的实验模型，尤其在鸡胚发育早期绒毛尿囊膜阶段，因其血管丰富，是天然的免疫缺陷宿主，是病理学、药理学和肿瘤学等研究领域的理想实验模型。作者简述了发育早期的鸡胚组织结构，并介绍了鸡胚绒毛尿囊膜在肿瘤研究、血管生成、器官移植、烧伤等疾病机理研究中的应用，以及在鸡胚病理模型基础上进行的抗肿瘤药物筛选的应用。重点介绍了鸡胚及禽类细胞系在病毒繁殖和疫苗生产、治疗性蛋白和单抗生产方面的应用研究进展。多种人源病毒、禽源病毒、支原体等可在鸡胚及禽类细胞上增殖，并用于疫苗生产。作者对常用的禽类纤维原细胞和多能干细胞的发展和特点进行了阐述，并总结了商业化的禽类细胞系来源以及部分易感病毒。鸡胚表达系统能够在目的蛋白特定位点产生人源化糖基，减少目的蛋白对人的过敏反应，且禽蛋廉价易得，可作为生产人用单克隆抗体和治疗性蛋白的合适供体。作者介绍了鸡胚及禽类细胞系在生物医药领域应用的最新进展，并对鸡胚作为动物模型在未来的应用进行了展望。     

鸡全胚成纤维细胞在鸡痘细胞活疫苗生产中的应用

采用鸡胚全部组织(全胚法)制备鸡胚成纤维单层细胞和采用鸡胚部分组织(常规法)制备鸡胚成纤维单层细胞生产了鸡痘细胞活疫苗,接毒后均产生大量的感染多核细胞和典型细胞融合性病变(胡椒粒样).鸡胚效价检测表明,鸡痘细胞苗和鸡痘组织苗均可引起鸡胚绒毛尿囊膜水肿、增厚或痘斑,但鸡痘细胞苗产生痘斑数量明显高于鸡痘组织苗.鸡体刺种结果表明,鸡痘细胞苗诱发的免疫反应(发痘)好于鸡痘组织苗;与常规法相比,全胚成纤维单层细胞制备方法可提高鸡胚利用率2倍以上.全胚法也可应用于制备其他鸡成纤维细胞疫苗.     

THE ISOLATION OF LENTOGENIC STRAINS OF NEWCASTLE DISEASE VIRUS IN AUSTRALIA

SUMMARY Twelve isolations of Newcastle disease virus were made from 77 clinical samples from chickens with conjunctivitis, respiratory disease, proventriculitis and bursal atrophy. Nine of the Isolations were made from chickens with conjunctivitis. The viruses were identified as Newcastle disease virus by inhibition of their haemagglutinins with specific antiserum to Newcastle disease virus. The viruses failed to kill chicken embryos after inoculation into the allantoic cavity and they were judged to be lentogenic strains. There was no evidence that the Newcastle disease viruses were responsible for any of the clinical conditions from which they were isolated. The presence of other agents in 10 of the samples was indicated by reduced production of haemagglutinin in allantoic fluids of infected embryos, by deaths of infected embryos, by the production of cytopathic changes in avian cell cultures and by electron microscopy. Three isolations of infectious bronchitis virus, 2 of avian adenovirus and one of avian reovirus were made. Other samples were suspected of containing infectious bronchitis virus and mycoplasmas, but these were not isolated. The Newcastle disease viruses failed to produce plaques in chicken embryo fibroblast cell cultures and they were separated from the contaminating agents by haemagglutination and elution followed by passage at terminal dilution in chick embryos. No Newcastle disease virus was isolated from 60 caecal tonsils and 60 lung samples from 9-week-old broiler chickens. Eight lung samples yielded mycoplasmas that caused haemadsorption in chicken cell cultures. The mycoplasmas were probably Mycoplasma gallisepticum.     

Infectious tenosynovitis in broilers and broiler breeders in Egypt

Two infectious tenosynovitis-producing viruses were isolated from tendon sheaths and synovial fluids of 59 broilers and 15 broiler breeders obtained from different flocks in Egypt during June to October 1983. The viruses grew well on the chorioallantoic membrane of developing chicken embryos, produced small localized white pock lesions with oedematous swellings at the inoculation sites and death of most of the embryos 72 to 96 hours post-inoculation. They also induced cytopathic effect in chicken embryo rough, Vero and MS cell lines. The viruses were neutralized by reovirus S1133 antiserum, both in tissue culture and. on the chorioallantoic membrane. Inoculation of the viruses into 2-day-old broiler chicks via the foot pad, intramuscular and oral routes reproduced the disease with the development of characteristic clinical, pathological and serological responses. The infection was transmitted to in-contact control chicks. This is the first report of the disease and of the isolation and identification of the causative virus in Egypt.     

Infectious tenosynovitis in broilers and broiler breeders in Egypt

Two infectious tenosynovitis-producing viruses were isolated from tendon sheaths and synovial fluids of 59 broilers and 15 broiler breeders obtained from different flocks in Egypt during June to October 1983. The viruses grew well on the chorioallantoic membrane of developing chicken embryos, produced small localized white pock lesions with oedematous swellings at the inoculation sites and death of most of the embryos 72 to 96 hours post-inoculation. They also induced cytopathic effect in chicken embryo rough, Vero and MS cell lines. The viruses were neutralized by reovirus S1133 antiserum, both in tissue culture and on the chorioallantoic membrane. Inoculation of the viruses into 2-day-old broiler chicks via the foot pad, intramuscular and oral routes reproduced the disease with the development of characteristic clinical, pathological and serological responses. The infection was transmitted to in-contact control chicks. This is the first report of the disease and of the isolation and identification of the causative virus in Eqypt.     

新城疫、禽流感病毒同胚接种的研究

根据新城疫病毒、禽流感病毒两种病毒均能在鸡胚中繁殖的特点,将两种病毒以最适稀释比例稀释后,分别接种于9—11日龄的鸡胚尿囊腔,同时进行两种病毒同胚培养,并利用血凝试验进行病毒效价的测定。结果表明,在同一鸡胚中两种病毒均能较好地增殖,病毒间干扰现象不明显。本试验在增强疫苗的免疫原性和进一步阐明病毒的增殖机理方面具有重要意义。     

The method of chicken whole embryo culture using the eggshell windowing,surrogate eggshell and ex ovo culture system

1. The unique accessibility of the avian embryo have made them an ideal model for the study of development and genome editing. Chicken whole embryo culture has provided important insights into toxicity tests, gene manipulation, clarifying gene functions, cell transplantation and cell tracking.

2. A simple technique for chicken manipulation is eggshell windowing, without or with seal, the latter having demonstrated some improvement in hatching rates.

3. Likewise, a surrogate eggshell system provides an accessible model for manipulation during chicken and quail development, with a higher hatchability compared to the simple windowing method.

4. The development of the chicken ex ovo culture systems in a synthetic environment as an efficient technique for imaging and microsurgery applications has enabled the study of important events of live chicken embryos at a specific time point.

5. This short review illustrates recent applications of well-designed whole embryo culture systems as a robust model for research into numerous biological mechanism, drug discovery, gene manipulating and production of functional proteins.     

鸡痘病毒诱发细胞融合现象的观察

鸡痘病毒在鸡胚绒毛作囊膜及鸡胚成纤维细胞上交替传代后，用本试验提供的条件，可以明显诱发鸡胚成纤维细胞的融合，形成多量多核细胞。经鸡胚及实验鸡回归试验证实，这种细胞融合现象确系鸡痘病毒所为。     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

Inhibition of in vitro lymphocyte blastogenesis by chicken alpha-fetoprotein

Chicken alpha-fetoprotein (ch-AFP), purified from fetal chicken serum and embryo extracts, respectively, was examined for its immunomodulatory effect in vitro. Significant (P less than 0.05) suppression of the allogeneic mixed lymphocyte reaction (MLR) was observed, when these preparations were added to one-way mixed lymphocyte cultures (MLC) in quantities of 62.5-1000 micrograms/ml. Suppression of the MLR was depending on the presence of ch-AFP for at least 16 h after initiation of the MLC, suggesting that this fetal protein was acting mainly in the early phase of lymphoblastogenesis. Serum of chicken embryos (12th and 15th day of incubation), day-old chickens, and of 10-week-old chickens of four different inbred lines were also found to exert suppression of the MLR. From these data, it is hypothesized that ch-AFP plays an immunoregulatory role by maintaining a certain stage of self tolerance during differentiation of the avian immune system.     

Chicken primary enterocytes: inhibition of Eimeria tenella replication after activation with crude interferon-gamma supernatants

A reproducible and original method for the preparation of chicken intestine epithelial cells from 18-day-old embryos for long-term culture was obtained by using a mechanical isolation procedure, as opposed to previous isolation methods using relatively high concentrations of trypsin, collagenase, or EDTA. Chicken intestine epithelial cells typically expressed keratin and chicken E-cadherin, in contrast to chicken embryo fibroblasts, and they increased cell surface MHC II after activation with crude IFN-gamma containing supernatants, obtained from chicken spleen cells stimulated with concanavalin A or transformed by reticuloendotheliosis virus. Eimeria tenella was shown to be able to develop until the schizont stage after 46 hr of culture in these chicken intestinal epithelial cells, but it was not able to develop further. However, activation with IFN-gamma containing supernatants resulted in strong inhibition of parasite replication, as shown by incorporation of [3H]uracil. Thus, chicken enterocytes, which are the specific target of Eimeria development in vivo, could be considered as potential local effector cells involved in the protective response against this parasite.     

Biological characterisation of an Indian isolate of egg drop syndrome-76 virus

An isolate of egg drop syndrome-76 virus replicated best in primary chicken embryo liver cells and less well in duck embryo liver cells, duck embryo fibroblast cells and chicken embryo kidney cells. The cytopathic effect in chicken embryo liver cells was marked by the presence of round and refractile cells and detachment of cells from the glass surface. The intranuclear eosinophilic inclusion bodies were observed by 24 to 48 hours after infection. No virus multiplication was observed in primary quail embryo fibroblast cells, chicken embryo fibroblast cells or mammalian cells like Vero, BHK-21 and MDBK. Duck embryos supported the maximum growth of the virus, with allantoic fluid having the highest haemagglutinin titre, followed in order by chorioallantoic membrane, skin and internal organs. Chicken and quail embryos did not support the growth of the virus.     

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU‐23 embryonic culture medium was used for a control group, while NCSU‐23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4‐ and 8‐cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2‐, 4‐, 8‐cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8‐cell versus the 4‐cell stage in control group as well as in the 8‐cell Ber group versus the 8‐cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber‐regulated miRNAs at the 8‐cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8‐cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.     

中草药抑制H_5亚型禽流感病毒对鸡胚毒性的研究

根据中医利用解表、清瘟、凉血止血的中草药防治人类流感的原理,选取金银花、北板蓝根、贯叶连翘、双黄连、贯众等五种中草药所制成的制剂进行H5亚型禽流感病毒感染鸡胚后毒力作用的抑制实验。首先对鸡胚接种禽流感H5亚型病毒,然后对感染鸡胚分别注射上述几种中药制剂,再抽取胚液进行HA和HI实验,将其中对鸡源H5-AIV有抑制作用的中草药挑选出来,测定中草药对禽流感病毒的治疗指数(Therapeutic index,TI)。然后采用寇氏法计算药物半数有效量ED50。结果表明,金银花、北板蓝根、贯叶连翘、双黄连、贯众五种中草药制剂在对H5亚型禽流感病毒的鸡胚毒性方面均有较好的抑制作用。     

An Avian Model for Comparative Studies of Insulin Teratogenicity

This study was designed to explore the effects of purified insulin during early stages of chick embryo development, and to search for variations between different molecular structures of the hormone. Chicken embryos were treated in ovo with a single dose of insulin (porcine or bovine), in only one stage of development between day 0 and day 9. Two susceptible periods were found. The earliest period (day 0 to day 3), characterized by abnormalities in the caudal vertebrae and a high mortality rate, was followed by a period with a different set of malformations, a syndrome classified as achondroplasia. The rate of achondro-plastic embryos was significantly higher with porcine rather than with bovine insulin. Paradoxically, insulin at physiological doses has stimulatory effects in growth and development but, in contrast, has inhibitory effects at higher doses. The precise signalling cascade of events in the target cells is still unclear. The possible interpretations of our results are discussed. The similarity between the insulin-induced abnormalities in the chicken embryos and the caudal regression syndrome, the most common malformation found in infants of diabetic women, suggests a common mechanism. This circumstance offers the chicken embryos as an excellent in vivo model for research on the mechanism of action of insulin during normal and abnormal development.     

禽用活疫苗外源病毒污染情况的调查研究

为对禽用活疫苗污染外源病毒的情况进行彻底摸底调查,抽取了目前使用量大、覆盖面广的禽用活疫苗重点品种共8种28批,涉及18家国内生产企业、7家国外企业,疫苗生产用SPF鸡胚来自国内8家企业。按照《中国兽药典》2010年版三部鸡检查法和《欧洲药典》对抽检的禽病活疫苗进行了外源病毒污染和禽腺病毒污染检验。结果显示所抽检的禽用活疫苗均无外源病毒污染和禽腺病毒污染,表明我国禽用活疫苗的质量控制和安全性良好。     

鸡胚大头开孔原壳体外培养方法的建立

通过原壳体外培养和抽蛋清的方法,就胚龄、蛋清抽取量对鸡胚发育的影响进行了研究。结果表明:①0日龄鸡胚抽取1mL、3mL和6mL蛋清后,其孵化率分别为38.6%,30.35%和8.6%。抽取蛋清1mL组(P<0.01)和3mL组种蛋(P<0.05)的孵化率明显优于抽取6mL组。②对3日龄鸡胚,抽取9mL蛋清后,鸡胚的发育受到的影响最大,其孵化率仅为33.3%,明显低于3mL和6mL组(P<0.01);而抽取3mL和6mL蛋清对3日龄鸡胚的影响较小,其孵化率仍高达74.3%和76.5%。③抽取6mL蛋清并在大头顶端开直径为1.0cm的圆孔进行鸡胚原壳体外培养获得成功,3日龄鸡胚的孵化率为18.5%。     

鸡早期胚胎精原细胞和睾丸发生发育关系的研究

采用连续切片技术，研究了15～45期(孵化50h～19d)鸡早期胚胎发育过程中精原细胞与睾丸发生发育的关系。结果显示：孵化第22～28期(第3．5～5天)，原始生殖腺内的原始生殖细胞(PGCs)胞质内的糖原颗粒开始分解；第29期(孵化第6天)，鸡胚性腺开始分化，PGCs的糖原颗粒进一步分解；第31期(孵化第7天)，PGCs内的糖原颗粒完全消失。在雄性，性腺开始显示睾丸特征，PGCs分化为精原细胞；第34期(孵化第8天)，睾丸内曲精细管索开始形成，呈实心状，精原细胞位于其中；第35～37期(孵化第9～11天)，曲精细管索数量逐渐增加，索内精原细胞体积较大，呈圆形单层排列，且已分化出支持细胞，但数量不多，形态不易分辨，间质内有少量间质细胞；第38～40期(孵化第12～14天)，可见到典型的曲精细管，精原细胞数量增加，支持细胞亦增多；曲精细管间的间质细胞成群分布。第40～45期(孵化第16～19天)，两侧睾丸大小略有差异，右侧稍大于左侧，精原细胞数量明显增多，呈葡萄串状分布在曲精细管的中央；曲精细管的管腔已经形成，精原细胞已分层排列。