牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

从牦牛输卵管壶腹部获取上皮细胞,用抽吸法从牦牛卵泡中获取颗粒细胞,对牦牛输卵管上皮细胞和卵泡颗粒细胞进行体外培养和传代的培养,观察培养过程中2种细胞原代和传代培养的生长方式及形态特点。两者培养144~216 h可形成融合的单层细胞,其传代密度保持105~106/mL为宜。     

Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in thi pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in the pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

Development of Purkinje cells in the ovine brain

Purkinje cells are involved in many vital functions within the body. Twenty ovine fetuses ranging from 2 to 5 months of gestation, two lambs in the first week after birth and three adult sheep were studied. Sections of the cerebellum were stained with haematoxylin and eosin, cresyl violet and Klüver-Barrera. This study indicates that Purkinje cells began to appear after the 15(th) week of gestation. There were varying degrees of development of Purkinje cells in different zones of the cerebellum. Our findings in sheep fetuses suggest that the maturation of Purkinje cells starts in the caudal regions of the cerebellum and that the process begins in the vermis before it does in the cerebellar hemispheres. The alignment of Purkinje cells was found to be very regular in the caudal regions of the cerebellum. A partial absence of Purkinje cells in the rostral regions of the cerebellum was observed in both sheep fetuses and adult sheep. In the first post-natal week, some ectopic Purkinje cells were found in the white matter of the cerebellum.     

Polyostotic hyperostosis associated with oviductal tumor in a cockatiel

A 10-year-old female cockatiel in a thin and depressed condition and with a 1 X 2-cm soft abdominal swelling was hospitalized. Radiography revealed a well-circumscribed mass containing punctate areas of mineralization, displacing the gizzard cranially in the caudodorsal portion of the abdomen. There was bilateral, symmetric, generalized, increased medullary opacity of the radius, ulna, carpometacarpus, femur, tibiotarsus, and tarsometatarsus. These findings were compatible with polyostotic hyperostosis secondary to an estrogen-secreting tumor. Euthanasia and necropsy were requested. A small area of neoplastic cells in the oviduct was identified histologically, but most of the tissue mass consisted of necrotic cellular debris. There was no metastasis. Sagittal secretions of decalcified radiopaque bones revealed up to 60% filling of medullary cavities with bone spicules. Although clinical and radiographic findings were compatible with an estrogen-secreting tumor, osteopetrosis, metastatic neoplasia, hypertrophic osteopathy, and metabolic bone disease were included in the initial differential diagnosis. The absence of metastasis and the confinement of the tumor within the oviduct would have made surgical removal a possible approach to treatment.     

羊梭菌病多联灭活疫苗生产用厌氧培养基的研究

通过多次培养试验,研制成功了新的厌氧培养基.该培养基用于羊梭菌病多联灭活疫苗中的产气荚膜梭菌(B型、D型)和腐败梭菌的培养,细菌的产毒能力和免疫原性均达到了<中华人民共和国兽用生物制品规程>(简称<规程>)的要求,配制的疫苗成品检验全部合格.该培养基与<规程>提供的肉肝胃酶消化汤培养基相比,配制方法简单,节约人力和时间,所用试剂均可从市场购买,所需原料成本低廉,仅为肉肝胃酶消化汤培养基的21%～27%.     

Growth of differentiated ovine tracheal epithelial cells in vitro

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.     

Maedi-visna virus infection of ovine mammary epithelial cells

The aim of this work was to perform a complete study of maedi-visna virus (MVV) infected mammary glands of naturally-infected sheep, and to determine if cells other than macrophages undergo a productive viral infection in this organ. Fifteen seropositive and two seronegative ewes were selected from MVV-infected flocks on the basis of clinical indurative mastitis and three sheep from an MVV-free flock. Within the mammary gland, MVV-positive cells were located by immunohistochemistry in the stroma and the epithelial alveolar barrier, most likely the ovine mammary epithelial cells (OMEC) of the acini. In situ hybridization confirmed these findings. Ultrastructural studies showed the presence of lentivirus-like particles budding off the cell surface in the alveolar barrier and also free in the acinar lumen. The presence of mammary histopathological lesions and MVV together with clear indications of productive infection (demonstration of a cytopathic effect in OMEC cultures and infection of co-cultures) were observed in the 15 seropositive and one of the seronegative sheep from the infected flock. These findings demonstrate that the OMEC were infected in vivo and probably underwent productive infection when studied ex-vivo. The OMEC of MVV-free sheep, which had subsequently been infected in vitro with MVV, also showed productive infection when challenged in vitro, confirming the replication of MVV in OMEC in vitro. The presence of MVV-infected OMEC in the mammary gland from infected animals, the productive infection in these OMEC and the release of lentiviral particles to the acinar lumen may have relevance in the pathogenesis and transmission of MVV infection.     

感染绵羊痘病毒的绵羊睾丸细胞外泌体源性蛋白组学分析

外泌体是细胞主动分泌的纳米级囊泡,参与机体多种生理和病理学过程,具有重要的生物学功能。为了探究外泌体源性相关蛋白在绵羊痘病毒感染过程中的作用机制,本试验利用绵羊痘病毒感染绵羊睾丸细胞,分离外泌体,并对分离的外泌体源性蛋白进行质谱和蛋白组分析。将获得的原始数据与蛋白质数据库进行匹配和质检筛选,共鉴别出453种宿主细胞相关蛋白和6种绵羊痘病毒蛋白。GO通路分析表明,感染绵羊痘病毒的绵羊睾丸细胞外泌体源性蛋白参与复杂的细胞生理过程,主要包括免疫反应、内吞途径、代谢及生物调控等过程,在绵羊痘病毒的感染和扩散过程中起着重要作用。这有望为绵羊痘疫病的诊断试剂、新型疫苗及治疗药物的研发提供理论基础和依据。     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

Co-culture of in vitro fertilized bovine embryos with different cell monolayers.

The effects of different cell monolayers on in vitro development of early bovine embryos derived from in vitro maturation and fertilization were examined in this study. Early embryos (four to eight cells) were randomly allocated to bovine granulosa cell (GC), oviductal cell (OC), or uterine cell (UC) monolayers in Exp. 1 and to GC, skin cell (SC; from 10-d-old chicken embryos), testicular cell (TC; from 10-d-old mouse), and liver cell (LC; from 10-d-old chicken embryos) monolayers in Exp. 2, and cultured for 6 d at 38.6 degrees C in a humidified atmosphere of 5% CO2 in air. The culture medium was 12.5 mM HEPES TCM 199 supplemented with 1% calf serum and 1 mM sodium pyruvate. In Exp. 1, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, OC, and UC monolayers was 26.9 (28/104), 37.5 (39/104), and 39.2 (40/102), respectively. In Exp. 2, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, SC, TC, and LC monolayers was 53.3 (40/75), 42.9 (33/77), 49.3 (37/75), and 44.3 (35/79), respectively. There were no significant differences in development among groups in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

EP2受体和FP受体对奶牛输卵管上皮细胞输卵管蛋白的影响

采用免疫荧光法,使用角蛋白鉴定奶牛输卵管上皮细胞;采用荧光定量PCR法检测奶牛输卵管上皮细胞输卵管蛋白mRNA的表达;采用Wsetern blot法检测了奶牛输卵管上皮细胞输卵管蛋白的表达。结果显示:加入butaprost(EP2受体激动剂,10~(-6) mol/L)能促进输卵管蛋白的表达,加入fluprostenol(FP受体激动剂,10~(-6) mol/L)能抑制输卵管蛋白的表达。结果表明:PGE2在奶牛输卵管上通过激活EP2受体上调输卵管蛋白的表达,PGF2α通过激活FP受体下调输卵管蛋白的表达。     

雌激素与前列腺素受体对奶牛输卵管上皮细胞输卵管蛋白合成和分泌的影响

旨在研究雌激素与前列腺素受体对奶牛输卵管上皮细胞输卵管蛋白合成与分泌的调控作用。用荧光定量PCR检测了奶牛输卵管上皮细胞输卵管蛋白mRNA的表达;采用Western blot检测了奶牛输卵管上皮细胞输卵管蛋白的表达。同时,加入雌激素(10~(-10)mol/L)和butaprost(PE受体激动剂,10~(-6)mol/L),雌激素(10~(-10)mol/L)和fluprostenol(PF受体激动剂,10-6mol/L),分别观察对输卵管蛋白表达的影响。结果表明:雌激素与PGE_2受体对奶牛输卵管上皮细胞输卵管蛋白的分泌调控具有协同作用;雌激素与PGF_(2α)受体对奶牛输卵管上皮细胞输卵管蛋白的分泌调控具有间接拮抗作用。     

Addition of alpha-tocopherol to culture medium improves the quality and cryosurvival of nuclear-transferred ovine embryos

The aim of this study was to reconstruct and cryopreserve somatic cell nuclear transferred (SCNT) ovine embryos and evaluate the effect of alpha-tocopherol on blastocyst development and subsequent cryosurvival of the SCNT embryos. The alpha-tocopherol (100 microg/ml) was added into culture medium for the SCNT embryos, the yield and total cell numbers of blastocysts were determined and the apoptosis incidences of the blastocysts were evaluated using the TUNEL assay. The blastocysts from the alpha-tocopherol and untreated groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. The results showed that there were no significant differences in blastocyst yield (26.3 vs. 22.3%) and total cell number (68.2 vs. 64.3) between the alpha-tocopherol and untreated groups. However, addition of alpha-tocopherol to the culture medium significantly decreased the apoptotic cell number (3.4 vs. 5.5) and significantly increased the cryosurvival of SCNT blastocysts (66.8 vs. 50.7%). In conclusion, addition of alpha-tocopherol to SCNT embryo culture medium was beneficial for improving embryo quality by decreasing the apoptotic blastocyst cell number and improving the tolerance of the embryos to cryopreservation.