利用鸡胚培养骨骼肌细胞的研究

细胞培养广泛地应用于病毒学,免疫学,遗传学,分化及发育等许多领域.近年来病毒学中很多发展与能在培养细胞中生长病毒的技术有关.利用细胞培养技术已经生产出多种生物制品,随着病毒学等研究工作的发展,鸡骨骼肌细胞培养工作越来越受到重视,本试验采用胰酶消化法培养了大量优质的鸡骨骼肌细胞,并进行了冻存与复苏,建立了一套较完备,快速的鸡骨骼肌细胞培养方法.     

鸽胚胎嗉囊成纤维细胞的分离培养及鉴定

为探究鸽嗉囊成纤维细胞的体外分离及培养方法,研究选择17日胚龄的鸽嗉囊组织,采用胰酶消化法和组织块贴壁培养法进行原代细胞分离,通过差速贴壁法纯化成纤维细胞.通过细胞形态观察、生长曲线测定和免疫荧光反应对细胞类型进行鉴定.结果 表明,采用胰酶消化法分离培养30 h后细胞开始生长,采用组织块贴壁培养法的细胞培养72 h后开...     

利用鸡胚培养骨骼肌细胞的研究

细胞培养广泛地应用于病毒学,免疫学,遗传学,分化及发育等许多领域。近年来病毒学中很多发展与能在培养细胞中生长病毒的技术有关。利用细胞培养技术已经生产出多种生物制品,随着病毒学等研究工作的发展,鸡骨骼肌细胞培养工作越来越受到重视,本试验采用胰酶消化法培养了大量优质的鸡骨骼肌细胞,并进行了冻存与复苏,建立了一套较完备,快速的鸡骨骼肌细胞培养方法。     

微载体细胞培养技术在兽医领域的应用

人们不但已知活细胞是构成所有活的有机体的基本单位,而且对其结构、功能、生命活动,以及在机体内不同细胞间构成的细胞社会、各种细胞与细胞周围环境间的关系等方面,进行了不同水平、不同层次、不同深度的探索,并随着科学技术水平的不断提高而向纵深发展.1885年Roux最先开始尝试使组织脱离机体而生存.1907年Harrison开始把组织培养作为一种方法,用于研究离体动物细胞.组织培养是一个通用名词,其实际内容可概括为三个不同概念,也就是包括有三种主要方法:(1)器官培养,培养物保持全部或部分体内组织结构,培养于液体与气体的交界;(2)原代外植块培养,将一小片组织放在玻璃的或塑料培养器皿与液体的交界处,待组织块粘着后,沿底平面移动生长;(3)细胞培养,将原代外植块生长晕用机械法或酸法分离细胞,作成细胞悬液,再培养于固体基质上,或单层细胞生长,或在培养液中呈悬浮状态培养.微载体细胞培养技术的诞生,为那些在悬浮培养条件下受限制的细胞可以大规模培养,把细胞培养提到崭新的高度.微载体细胞培养技术使细胞培养量在单位体积中可增加数倍甚至10倍以上.     

动物细胞规模化培养及其在兽用疫苗生产中的应用

哺乳动物细胞大规模培养技术已经广泛应用于生产各种生物制品,也广泛应用于各种兽用疫苗的生产.细胞培养生产兽用疫苗不但能够降低成本,而且提高产品质量.细胞培养生产兽用疫苗已经成为各个生物制品公司关注的焦点.凡是影响细胞大规模培养的因素,都直接或者间接的影响疫苗质量.论文针对哺乳动物细胞大规模培养相关影响因素、相关技术和在兽用疫苗中的应用进行概述.     

简单、实用、高效的细胞培养技术

通过近几年的实践,笔者对我国兽医生物制品生产中传统的细胞培养工艺进行了一些改进,包括用洗洁精代替其他洗液,用自行研制的营养液代替常规营养液用于细胞培养,改变培养时的转速,改变细胞消化液,改进细胞的保存方法.这些方法均成功应用于生产,取得了良好的效果.     

半原位灌流法分离成年鸡肝细胞及原代培养

采用半原位灌流法,对40~50日龄的海蓝公鸡进行肝细胞分离,采用台盼蓝拒染法测定细胞总数并计算肝细胞实时存活率,肝细胞以5×105m L的密度接种于6孔细胞培养板,每孔3 m L。在CO2培养箱中培养,观察细胞形态,测定不同培养阶段细胞培养上清液中LDH活力。结果显示每只鸡肝脏分离获得的细胞产量为(4.1±0.2)×108个,肝细胞实时存活率为(95±1)%,培养48 h至144h,细胞上清液中LDH活力在低水平下保持稳定,细胞可维持良好的生长状态达7 d以上。显示该方法可用于分离培养成年鸡肝细胞,分离培养的鸡肝脏细胞可用于研究细胞代谢、基因表达和肝细胞毒性。     

小鼠成肌细胞培养方法的建立与优化

通过对小鼠成肌细胞C2C12细胞培养,优化细胞的培养方法和培养条件,提出了培养C2C12细胞的最佳条件.获得大量贴壁培养的小鼠成肌细胞C2C12,将广泛地应用于生物医学、肌肉再生临床医学、动物肉质性状相关研究及多种与骨骼肌相关的因子机制研究.     

不同生长时期牛血清对细胞培养效果的影响

[目的]为了检测不同生长时期的牛血清的细胞培养效果,从而在今后的实验中更加合理地选择牛血清进行细胞培养。[方法]实验采用两组采自不同年龄段的牛血清对Sp2/0—Ag14细胞进行培养,并对细胞生长的细胞数量、细胞形态等定期进行测定及观察并绘制出细胞生长曲线,计算出细胞倍增时间,以比较两组牛血清细胞培养的效果。实验分为两组:一组为3月龄采血的牛血清(A组),另一组为14日龄采血的牛血清(B组)。[结论]结果表明,应用两组牛血清培养的Sp2/0—Ag14细胞的培养倍增时间分别为A组牛血清17.8h和B组牛血清17.1h;表明B组牛血清的细胞培养效果优于A组牛血清。     

犬子宫内膜上皮细胞和基质细胞的分离培养与鉴定

为了探究犬子宫内膜细胞的培养方法,采用组织块培养法和酶消化培养法进行细胞培养并使用倒置显微镜观察细胞的形态结构和生长状态,对细胞进行免疫组化鉴定。结果表明,组织块培养法需要1周左右培养出犬子宫内膜上皮细胞,且细胞不易纯化;酶消化培养法节省时间,获得的细胞活力强、纯度高。综上所述酶消化培养法更适于培养犬子宫内膜细胞,为后期细胞水平研究奠定基础。     

Histopathological and Immunohistochemical Characterization of Spontaneously Occurring Uterine Deciduomas in Young Adult Rats

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.     

Embryo resorption following administration of steroidal compounds to rats in mid pregnancy.

In the course of experiments on the effects of anabolic steroids on the myocardium of rat conceptuses, we found that subcutaneous implantation of 10 mg of estradiol, Dianabol or testosterone to rats in mid pregnancy, resulted in embryo resorption. Placental tissue was identified only in estradiol-treated rats which also demonstrated a large amount of serosanguineous fluid that dilated the horns considerably. The yellow nodules of placental attachment sites were represented histologically by cellular and vascular proliferations between the inner and outer layers of the myometrium. The nodular aggregates of cells had variable features according to the steroid administered. Neither decidual cells nor metrial glands that are reported to be the constituents of placental attachment sites were seen in our material. We conclude that anabolic steroids are potent agents for embryo resorption, and that the cells in the nodules of placental attachment sites are likely to be derived from the myometrium.     

Malignant mixed mullerian tumor in a rabbit (Oryctolagus cuniculus): case report with immunohistochemistry

A rabbit (Oryctolagus cuniculus) with a homologous malignant mixed müllerian tumor (MMMT) of the uterus with decidualization in the sarcomatous components is described. On histologic examination, the neoplasm was characterized by a carcinomatous and a sarcomatous component with invasion of the myometrium. The epithelial component was a well-differentiated carcinoma, and the nonepithelial component contained large amounts of intracytoplasmic glycogen. The changes in stromal cells were morphologically similar to changes usually found in decidual cells in the pregnant uterus or in deciduosarcomas in rabbits. Results of immunohistochemical analysis indicated that the epithelial components stained positive with cytokeratin (CK7, AE1/3) and the decidual-stromal cells stained positive for vimentin, but did not stain with alpha-SMA, actin, and desmin. This case fulfills all the criteria of an MMMT in having a carcinomatous and a sarcomatous component, but differs from cases of MMMT in women in that the sarcomatous component had decidualized. To the authors' knowledge, this is the first report of a malignant mixed müllerian tumor in rabbits.     

Early maternal changes contributing to the formation of the chorioallantoic and yolk sac placentas in rat: a morphological study

In order to determine the maternal changes contributing to the formation of the chorioallantoic and yolk-sac placentas, rat gestation sites were examined by light and electron microscopy on days 7 through 10 of pregnancy. On day 7, the implantation chamber showed different compartments and contained the blastocyst in the antimesometrial chamber. The epithelial lining of the implantation chamber disappeared at the antimesometrial chamber, transformed into disintegrated cells in the mesometrial chamber, and showed signs of the programmed cell death in the decidual crypt. On day 8, the mesometrial chamber lumen contained red blood cells and it was continuous with subepithelial sinusoids. The endothelial cells lining the mesometrial sinusoids also showed some characteristics of the sprouting type angiogenesis such as hypertrophy and cell proliferation. While the yolk-sac placental circulation was more obvious with participation of the giant trophoblasts at the antimesometrial pole of the conceptus on day 9, the antimesometrial cells showed autophagic degeneration after the formation of the chorioallantoic placenta on day 10. The contribution of the regional cell death and angiogenesis to form both of the two placentas are discussed.     

膜联蛋白A8在小鼠早期妊娠子宫中表达研究

膜联蛋白A8(Annexin A8,ANXA8)是一种磷脂结合蛋白,与炎症反应、癌症的发生以及血管生成有密切联系。本实验旨在利用实时荧光定量PCR、原位杂交与免疫组织化学的方法研究ANXA8 mRNA与蛋白在小鼠早期妊娠和人工蜕膜子宫中的表达。原位杂交结果表明:ANXA8 mRNA在小鼠早期妊娠第1～4天子宫腔上皮和腺上皮有微弱表达,ANXA8 mRNA在妊娠第5、6天的初级蜕膜区与第7、8天的次级蜕膜区表达,并随妊娠进行逐渐增强;人工蜕膜化模型中ANXA8 mRNA表达在蜕膜区。实时荧光定量PCR证明:ANXA8 mRNA的表达量在早期妊娠模型中的第7、8天显著提高,人工蜕膜侧子宫与对照侧相比也显著提高。免疫组织化学结果表明:ANXA8蛋白与ANXA8 mRNA表达规律相似。体外分离培养小鼠子宫基质细胞,并诱导蜕膜化,实时荧光定量PCR结果表明ANXA8随着基质细胞的蜕膜化表达升高。以上体内和体外实验表明,ANXA8在小鼠子宫中的表达具有着床相关特异性,ANXA8参与小鼠子宫蜕膜化过程。     

Immunohistochemical detection of the orexin system in the placenta of cats

The aim of the present study was to investigate the presence and distribution of cells containing orexin A (OXA), and orexin type 1 and 2 receptors (OX1R and OX2R, respectively) in the feline placenta by means of immunohistochemical technique. OXA was identified in several decidual and syncytiotrophoblastic cells present in the lamellar portion of the placenta. In the same placental structures, few decidual and syncytiotrophoblastic cells showed the presence of OX1R-like immunoreactivity. Characteristically, immunopositivity for OX2R, but not for OX1R, was evidenced in the cells of the glandular layer. The orexinic system was not expressed in the uterine structures that were not engaged by the chorion. Our results provide the first evidence of the presence of a placental orexinic system in a mammalian species. Orexin A and both OX1R and OX2R are unequally distributed within the cat placenta. Local OXA production and the presence of specific receptors, differentially expressed in the placental structures of the cat, suggest that the orexinic system may participate in placental growth and development as well as in the regulation of its steroidogenic capacity via endocrine, paracrine and/or autocrine mechanisms.     

Involvement of stathmin in proliferation and differentiation of immortalized human endometrial stromal cells

Uterine endometrial stromal cells differentiate into decidual cells during the late secretory phase of the menstrual cycle and pregnancy. However, the biochemical mechanisms of decidualization have yet to be definitively elucidated. In the present study, we transfected primary human endometrial stromal cell with a temperature-sensitive mutant of simian virus 40 large T antigen and thereby established an immortalized stromal cell line (EtsT) in order to examine the role of stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, in stromal cell differentiation. When treated with the decidual stimulus dibutyryl-cAMP (db-cAMP) or forskolin, the fibroblastic cell-shaped EtsT cells transformed into large- and round-shaped cells and secreted large amounts of the decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Analysis of the stathmin protein levels in the db-cAMP- and forskolin-treated EtsT cells revealed that the total and phosphorylated protein levels dropped as decidualization progressed. Suppression of stathmin expression by transfection with small interfering RNA (siRNA) suppressed EtsT cell proliferation. It also abolished db-cAMP-induced PRL and IGFBP-1 mRNA expression and protein secretion. Thus, stathmin expression can be considered an integral factor regulating the initial stage of the process of human endometrial stromal cell differentiation.     

Characterization of a population of unique granular lymphocytes in a bitch deciduoma, using a panel of histo- and immunohistochemical markers

The ovaries and uterus were collected after ovariohysterectomy from a 16-month-old Labrador bitch in diestrus that never mated. Discrete swellings were found in the uterine horns, with the macroscopic appearance of normal early pregnancy. At histologic examination, the endometrium, devoid of any conceptus and chorion, showed a marked proliferation, on the basis of which a diagnosis of deciduoma was made. A remarkable population of stromal eosinophilic granular lymphocytes was present, especially in the axis of the endometrial folds. Periodic acid-Schiff and Dolichos biflorus-lectin histochemical reaction and a panel of 10 immunohistochemical markers were used to characterize eosinophilic granular cells. Our findings allowed us to compare these granular cells with the granulated decidual cells, whose presence was until now described only in primates, rodents, or a few other epitheliochorial species. On the basis of our results, the importance of eosinophilic granular cells in a decidualization process is hypothesized to occur also in the bitch.     

Immunohistochemical detection of cellular prion protein (PrPc) in the rat central nervous system

A simple conventional method of immunohistochemistry (i.e. fixing the frozen sections in cold methanol) was used to determine the immunolocalization of cellular prion protein (PrPc), with good results. In the rat cerebrum, the cytoplasm of neural cells in the cortex and corpus stratum, pia mater, membrane limitans gliae superficialis, choroid plexus and blood vessel wall were immunostained. The formation of network structures of immunostained neural and/or glial fibers in the cerebral cortex was also observed. These immunostained network structures of neural and/or glial fibers were also observed in cultured neural cells. The results suggest that fixation of frozen sections and cultured cells with cold methanol is a useful method for detecting the immunolocalization of PrPc and that PrPc exists in the various components of the central nervous system of the rat.     

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N‐acetyl glucosamine residues and non‐sialylated galactose and N‐acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N‐linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N‐linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo‐maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.