Invasive ductal carcinoma of the mammary gland in a mare

A 21-year-old thoroughbred mare had a 35 x 14 x 10 cm mass involving the mammary gland. Metastases were found in the kidneys, lungs, skeletal muscles, and regional lymph nodes. Histopathologic examination of the tumor revealed a ductal solid carcinoma with extensive intraductal and intralobular involvement and focal infiltration of the adjacent stroma. The intralobular neoplasms were divided into irregularly shaped islands and sheets of polygonal and spindle-shaped epithelial cells by thick or thin fibrous connective tissue bundles. The neoplastic cells had a small or moderate amount of cytoplasm that stained faintly with eosin and round or oval hyperchromatic nuclei. Immunohistochemically, the neoplastic cells were strongly positive for Lu-5, weakly positive for AE1/AE3, vimentin, and glial fibrillary acidic protein, and negative for cytokeratin 8, cytokeratin 14, alpha-smooth muscle actin, calponin, and S100. The neoplasm was diagnosed as an invasive ductal carcinoma of the mammary gland with multiple metastases.     

Malignant fibrous histiocytoma (giant cell type) associated with a malignant mixed tumor in the salivary gland of a dog

A 12-year-old male Boxer dog presented with a 5 x 5 x 7-cm partially encapsulated mass in the right mandibular salivary gland. Histologically, the mass was composed of neoplastic epithelial and mesenchymal cells. The mesenchymal component consisted of two cell populations arranged in different patterns: coalescing nodules of neoplastic mononuclear cells with rare osteoid and numerous osteoclastlike giant cells; and sheets of neoplastic spindle cells intermingled with neoplastic epithelial cells and containing osteoid and well-formed bone trabeculae lined by osteoblasts and few osteoclastlike giant cells. On the basis of these histological features, two malignant salivary tumors were diagnosed: a malignant fibrous histiocytoma (giant cell type) and a malignant mixed tumor. Immunohistochemical studies demonstrated keratin 5 and 8 expression by the neoplastic epithelial cells, indicating a probable salivary ductal origin, and vimentin expression by all mesenchymal elements, suggesting a fibroblastic line of differentiation.     

组织块再移法分离培养奶山羊乳腺上皮细胞

通过比较组织块贴壁培养法、组织块再移法、胰蛋白酶消化法、胶原酶Ⅱ消化法和胶原酶消化配合组织块贴壁法分离、培养、纯化山羊乳腺上皮细胞,绘制细胞生长曲线并计算细胞群体倍增时间,研究乳腺上皮细胞培养效果。结果显示,利用胶原酶消化配合组织块贴壁法不能获得正常生长的乳腺上皮细胞;利用组织块贴壁培养法、组织块再移法和胶原酶Ⅱ消化法获得大量的乳腺上皮细胞,所得到的上皮细胞生长曲线呈典型的＂S＂型,符合细胞生长的一般规律。组织块再移法不仅可快速获得大量纯化的乳腺上皮细胞,而且所得到的细胞经传代后,细胞群体倍增时间最短、增殖能力最强,表明,该法是获得山羊乳腺上皮细胞最适宜的方法。     

Localization of the sheep FcRn in the mammary gland

Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells.     

DNA measurement and immunohistochemical characterization of epithelial and mesenchymal cells in canine mixed mammary tumours: putative evidence for a common histogenesis.

DNA measurement by image cytometry, and a detailed immunohistochemical study using monoclonal antibodies directed against different human cytokeratin types, muscle-specific actin, vimentin and S100 protein were carried out on normal canine mammary tissue (n =4), benign canine mammary mixed tumours (n =20) and malignant canine mammary mixed tumours (n =13). The results showed that ductal and alveolar luminal cells in normal and neoplastic tissue were immunoreactive with CAM5.2 and AE1/AE3 antibodies recognizing human keratins.Basal/myoepithelial cells were clearly differentiated from ductal and alveolar epithelial cells, since the latter only expressed cytokeratins, whereas the former also expressed vimentin and muscle-specific actin. This immunohistochemical study showed that there is loss of expression of muscle-specific actin and cytokeratins in areas of myoepithelial proliferation, and enhanced expression of vimentin and S100 protein in proliferative areas with osseous and/or chondroid metaplasia. The ploidy studies revealed that 20% (4/20) of benign and 54% (7/13) of malignant mixed tumours of canine mammary gland were aneuploid and that the epithelial and myoepithelial components of the mixed tumours had identical DNA content.Our results reinforce the role of myoepithelial cells in mesenchymal metaplasia in mixed mammary tumours and suggest the possibility of a common origin of both components from a totipotential stem cell with capacity for divergent differentiation.     

猪乳腺细胞分离培养及EGFP基因转化

本研究旨在从猪乳腺组织中分离得到上皮细胞和成纤维细胞,并将EGFP基因导入这些细胞.利用乳腺细胞堵养体系从成年猪乳腺组织中分离培养上皮细胞和成纤维细胞,并利用脂质体介导转染技术将EGFP基因导入这些细胞.结果,从成年猪乳腺组织中成功分离培养出上皮细胞和成纤维细胞,获得转EGFP基因上皮细胞和成纤维细胞.上皮细胞呈短梭形或多角形,细胞之间紧密相靠,互相衔接,连接成片;细胞核呈圆形或椭圆形,核仁2～4枚,比较明显.成纤维细胞呈长梭形.结果表明,可以从猪乳腺组织中分离上皮细胞和成纤维细胞,EGFP可以在这些细胞中表达.     

Electron microscopic study of leucocytic infiltration of the mammary teat duct during infection with Staphylococcus aureus

Leucocytic response to Staphylococcus aureus infection was observed in the bovine mammary teat duct using transmission electron microscopy. Leucocytes migrating across the stratified squamous epithelium were observed in close association with areas colonised by cocci. Leucocytes gained access across the epithelium to the teat duct lumen by: passage as luminal cells desquamated; migration through degenerate cell cytoplasm; and penetration of cell junctions. The results provide evidence of marked leucocytic infiltration into ductal tissue which may participate in the cellular response to mastitis.     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。     

Choroid plexus carcinoma cells in the cerebrospinal fluid of a Staffordshire Bull Terrier

An 11-year-old female intact Staffordshire Bull Terrier was referred to the Queen's Veterinary School Hospital at the University of Cambridge with sudden onset of episodic behavioral changes, a mammary mass, and papilledema in the right eye. On physical examination the dog appeared depressed and had a head tilt to the right with anisocoria. Using magnetic resonance imaging, a broad-based lesion that obliterated the fourth ventricle was detected in the right brainstem. There was no evidence of pulmonary metastasis. Cerebrospinal fluid (CSF) was then obtained; fluid analysis showed an increased cell count (165 cells/μL, reference interval 0-7 cells/μL) and total protein (0.30 g/L, reference value <0.25 g/L). Cytologic evaluation revealed a population of atypical epithelial cells arranged in cohesive rafts and characterized by moderate to occasionally marked anisocytosis and anisokaryosis. The appearance was highly suspicious of a malignant epithelial neoplasm. The dog was euthanized and on postmortem examination an asymmetrical nonencapsulated cerebellar mass was found within the choroid plexus of the fourth ventricle with local extension into the cerebellopontine angle. Histologic sections of the cerebellar mass contained arborizing papillary structures covered by a single layer of atypical epithelial cells that showed local infiltration into the adjacent neuropil. The diagnosis was choroid plexus carcinoma. The atypical epithelial cells were negative for pancytokeratin and strongly positive for vimentin. The finding of clusters of choroid plexus epithelial cells in the CSF demonstrates the value of utilizing a relatively noninvasive diagnostic technique for diagnosis of choroid plexus tumors.     

Evidence of a Direct Role for Growth Hormone (GH) in Mammary Gland Proliferation and Lactation

A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in ductal and alveolar epithelial cells from rat and rabbit mammary glands by immunohistochemistry. Intense immunoreactivity was present in membrane, cytoplasm and some nuclei of epithelial cells during proliferation and lactation. Receptor expression decreased during weaning and was absent or weak in regressive mammary glands. Immunoreactivity was weak in ductal epithelial cells from virgin adult animals. Pronounced expression of GH receptor/binding protein was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the existence of a soluble form on the GH receptor, namely the growth hormone binding protein derived from the membrane receptor by cleavage. Primary localization of the receptor in proliferating and lactating epithelial cells suggests that the rat and rabbit mammary gland is a GH target tissue. This finding is in contradiction to both classical GH action and the somatomedin hypothesis and challenges the widely held view that GH has no direct influence on mammary growth and function.     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

Plate and collagen-gel cultures of normal canine mammary epithelial cells.

Plate and collagen gel (floating or embedded) cultures were made to study the cell morphology and growth characteristics of canine mammary epithelial cells in vitro. Hiratsuka's method was satisfactory for the separation of the epithelial cells from the fibroblasts in explants of canine mammary tissue. In the plate culture method, epithelial cells migrated out from the explants to form colonies. The characteristic features of epithelial colonies in this culture system were polygonal cells situated in the centre, elliptical cells in the outer region and large spindle-shaped cells, thought to be myoepithelial cells, at the periphery. These epithelial cells showed a pavement-like appearance and were positive for antikeratin antibody. On floating gels, the cells changed their shape dramatically from flattened to rounded when the gel was released from the glass dish. Cells embedded in collagen gels grew to form duct-like structures.     

小鼠乳腺发育泌乳过程中L型氨基酸转运载体1的表达研究

为阐明L型氨基酸转运载体1（L-type amino acid transporter 1,LAT1）的表达与乳腺发育和泌乳功能之间的关系,采用荧光定量RT-PCR技术和激光共聚焦显微技术对青春期、妊娠期、泌乳期和退化期小鼠乳腺中LAT1及其辅因子4F2抗原重链（4F2hc）表达含量和部位的变化进行研究。结果表明,青春期乳腺导管发育缓慢,LAT1和4F2hc在导管上皮细胞膜、肌上皮细胞膜及乳腺脂肪细胞膜上均低水平表达;妊娠期导管上皮细胞增殖分化加速,LAT1和4F2hc在乳腺小叶导管上皮细胞膜基底侧表达,表达水平上调;泌乳期乳蛋白合成和分泌旺盛,LAT1和4F2hc在腺泡上皮细胞膜的基底侧表达,表达量达到峰值;退化期乳腺组织功能性结构消退,乳腺对氨基酸的需求降低,LAT1和4F2hc的表达下降。提示,LAT1/4F2hc是小鼠乳腺组织中转运氨基酸的载体形式,LAT1和4F2hc的表达变化与乳腺发育、泌乳、退化的生理过程中氨基酸的需要量相关。     

山羊乳腺上皮细胞基因转染条件的优化

为了优化外源基因转入乳腺上皮细胞的条件,本研究采用组织块培养法,从山羊乳腺组织中分离并获得了纯化的乳腺上皮细胞,通过染色角蛋白和测定生长曲线对其进行了鉴定和检测。然后利用电穿孔法将PEGFPC1载体导入乳腺上皮细胞,分别比较了在不同电压(120、140、160、180、200V)、不同电击时长(5、10、15、20ms)以及2株细胞(GMEC1、GMEC2)的不同代数(第1代、第3代、第5代、第9代)条件下的转染效率,并利用优化条件对山羊乳腺上皮细胞进行人β-防御素3基因转染,经过G418筛选,最终获得单克隆细胞,同时对获得的单克隆阳性细胞进行体外诱导表达,并对诱导产物进行Western blotting鉴定。结果表明,不同遗传背景的细胞对转染效率的影响较小,而第1代培养的细胞转染效率要显著高于传代细胞,乳腺上皮细胞在电压180V、电击时长15ms、电击1次的条件下转染效率最高,优化条件下转染、筛选和扩增后获得稳定表达人β-防御素3蛋白的单克隆乳腺上皮细胞。研究工作为乳蛋白基因表达调控机制的研究及乳腺特异性表达载体的检测提供参考资料。     

Isolation,Culture and Identification of Buffalo Mammary Epithelial Cells from Milk

This study was aimed to set up a simple, economic and pure method to culture and identify buffalo mammary epithelial cell in vitro, which were collected from the fresh milk. The milk was collected in the late lactation period of the high yield milk buffalo, then it was mixed with the PBS in the ratio of 2:1 and centrifugated. A few suspernatants and the pellets were resuspended with the PBS when the milk fat were decanted, then recntrifuged. The final pellets and 1 mL of suspernatants were seeded in petri dish without fat, respectively. The isolated cells were mammary epithelial cells which were identified by cell morphology, immunofluorescence, growth curve, cell viability and RT-PCR. The buffalo mammary epithelial cells were successfully isolated from both the pellets and supernatants. The cells and impurities were less in the supernatants than that in the pellets. The cells were cobblestone-like without fibroblasts and most in cell division. In the post-confluent culture, mammary epithelial cells formed dome structures and layer-separated growth. The cells expressed cytokeratin 18 was identified by immunofluorescence which was the marker of mammary cells. The cell growth curve and cell survival rate were measured. The results showed that the buffalo mammary epithelial cells were activity and easy to attach. The mammary epithelial cells were expressed of milk protein and milk fat related genes detected by RT-PCR. The buffalo mammary epithelial cell line were successfully isolated and identified from fresh milk, which estabilished foundation for the study of the mechanism of lactation, the amelioration of the quality of milk and the improvement of milk yield of buffalo.     

水牛乳汁中乳腺上皮细胞的分离培养及鉴定

试验旨在从水牛（Bubalus bubalis）乳汁中分离培养水牛乳腺上皮细胞并对其进行鉴定,建立经济、便捷的水牛乳腺上皮细胞分离方法。通过无菌收集高产奶水牛泌乳后期乳汁,将其与PBS按2：1（V/V）混匀离心;去除上层乳脂,取带少量上清的沉淀,与PBS按上述比例混匀再次离心;将脂肪层去除干净,取沉淀上方1 mL上清及沉淀分别接种于细胞培养皿。通过细胞形态、免疫荧光、生长曲线测定、细胞接种活力、RT-PCR等对分离的细胞进行鉴定。结果表明,试验成功从水牛乳汁的上清和沉淀中分离获得了水牛乳腺上皮细胞,上清部分细胞和杂质较少;而沉淀部分则细胞和杂质较多。细胞呈典型鹅卵石样,无成纤维细胞,细胞活力好,多处于分裂期。当细胞增殖高度融合时,出现乳头状结构和分层生长现象。经免疫荧光鉴定,分离获得的水牛乳腺上皮细胞表达上皮细胞标志蛋白--细胞角蛋白18。水牛乳腺上皮细胞的生长曲线呈S型,符合一般生物学规律。细胞接种存活率的测定结果显示,细胞活力好、贴壁能力强。经RT-PCR检测,水牛乳腺上皮细胞表达乳蛋白及乳脂合成相关基因。从乳汁中分离水牛乳腺上皮细胞并对其进行鉴定,成功建立了水牛乳汁分离乳腺上皮细胞的方法,为研究水牛泌乳机制、改善乳品质、提高产奶量奠定基础。