牦牛乳腺上皮细胞的体外分离培养及鉴定

为提高牦牛乳腺上皮细胞体外培养成功率,实现高度可重复性,试验采用2.5 g/L(含0.5 g/L EDTA)胰酶和Ⅰ型胶原酶(1 mg/mL)分段消化牦牛乳腺组织块以分离细胞,原代培养时于DMEM/F12培养体系内添加氢化可的松(1 μg/mL)、表皮生长因子(50 ng/mL)及胰岛素-转铁蛋白-硒(5 μg/mL)...     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

山羊乳腺上皮细胞的生长特性

用组织块培养法获得山羊乳腺细胞原代培养物。根据山羊乳腺成纤维细胞与上皮细胞对胰蛋白酶的敏感性不同将二者分离纯化，对细胞生长特性进行了光镜观察。结果如下：细胞可形成闭合型细胞群和开放型细胞群。乳腺上皮细胞与成纤维细胞混生时，细胞之间形成许多腔状结构。纯化的乳腺上皮细胞通过单细胞悬浮后传代，部分细胞形成岛屿状聚集，部分细胞以贴壁的自由单个细胞散在形式存在。上皮细胞增殖可形成圆顶型结构，呈乳头状，称之为乳球体；可产生乳腺上皮细胞并分泌乳汁。山羊乳腺上皮细胞含不同的细胞类型．大多数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，呈蜂窝状；细胞核呈圆形或椭圆形．核仁2～4枚；部分细胞呈圆饼状，体积较大；出现部分长形细胞；接触抑制的上皮细胞形态不均一。纯化的山羊乳腺上皮细胞传代至第15代时其生长仍正常，经透射电镜观察发现细胞表面微绒毛极为发达，细胞质中线粒体和粗面内质网丰富，细胞质内有大量脂滴及小泡，表明第15代山羊乳腺上皮细胞增殖活力旺盛。染色体数目分析表明．该细胞系稳定，在离体培养条件下细胞不发生转化。山羊乳腺上皮细胞系的细胞染色体数目为60．染色体组型为2n=60。     

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.     

Nuclear cytomorphometry in feline mammary gland epithelial tumours

Stained cytological specimens from 35 feline mammary gland epithelial tumours (4 adenomas, 11 tubulopapillary carcinomas 13 solid carcinomas and 7 cribriform carcinomas) were analysed by computer-assisted nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear diameter (MND) and nuclear roundness (NR) were calculated. The study aimed to evaluate (1) the possibility of using nuclear cytomorphometry as an auxiliary diagnostic method to differentiate between benign and malignant feline mammary gland epithelial tumours, and (2) the prognostic value of nuclear morphometry in feline mammary carcinomas. The results indicated that MNA, MNP, MND and NR could be a useful adjunct in diagnosis but are not reliable prognostic indicators for feline mammary gland carcinomas.     

Mammary mucinous carcinoma in the cat

Mucinous carcinoma of the mammary gland is a rare tumor characterized by excessive mucin production. In human and canine pathology, the diagnosis of mucinous carcinoma is based on the demonstration of an epithelial phenotype of mucus-producing cells and periodic acid-Schiff (PAS)-diastase positivity of the mucin. The histologic and immunohistologic characteristics of feline mucinous mammary carcinoma were examined. Of 656 cases of feline mammary neoplasms and dysplasias, 3.2% were found to be mucin-producing tumors. Cytokeratin 19 (16 cases positive, 4 heterogenous, and 1 negative) and vimentin (15 cases positive, 2 heterogenous, and 4 negative) expression were examined, and the mucin produced was alcian blue positive. PAS-diastase staining was variable (38.1%). Based on these findings, mucinous mammary carcinoma in the cat varies significantly from the human and canine varieties and alcian blue is the prominent stain in the diagnosis of feline mucinous carcinoma.     

猪翻乳头的解剖组织学特征的研究与探讨

通过对正常乳头，翻乳头，副乳头及其乳房的组织样本进行解剖学观察，对照，发现正常乳头由乳基，乳头本组成；翻乳头由乳基，不明显的乳头体组成；副乳头只有小突起。对各类乳腺做成切片观察，对照，发现正常乳头和翻乳头均有完整的乳腺，乳管，副乳头的乳腺，乳管不发达，且很稀少。     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

乳腺分泌物中体细胞的研究进展

不同物种的乳腺分泌物中含有的细胞成分被称为体细胞,其中包括淋巴细胞、白细胞、巨噬细胞和上皮细胞。物种、乳腺感染情况、不同生理阶段和饲养管理条件等因素均会影响乳中的体细胞数量和细胞类型。近年来,乳中体细胞得到了人们的关注和深入研究,显示出广阔的应用前景。人们利用从初乳和常乳中得到的乳腺上皮细胞已经成功进行了乳腺细胞的原代培养和建立了乳腺细胞系,为乳生成、被动免疫转移和乳腺癌的研究提供了良好平台。体细胞中提取的RNA代表了乳腺组织的基因表达,因此为研究乳腺组织的基因表达提供了方便、良好的来源。     

果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及风味影响的研究

试验旨在研究果香味剂对奶牛乳腺上皮细胞培养液中乳成分含量及培养液风味的影响。试验选用自制的奶牛乳腺上皮细胞,试验设5个处理组,果香味剂的添加剂量分别为0、5、10、15和20μg/ml。结果表明:①当培养基中果香味剂添加量为15μg/ml时,乳腺上皮细胞培养液中总蛋白水平、甘油三酯及乳糖含量均达到最高,其中对脂肪含量影响显著(P<0.05),而对蛋白和乳糖含量影响不显著(P>0.05),之后呈下降趋势,说明果香味剂可能会诱导奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖分泌水平的升高;②运用PT/GC-MS分析乳腺培养液中的风味成分,与对照组相比,各试验组中除具有原有乳腺培养液的风味外,还有乙酸异丁酯、乙酸异戊酯、丙酸异戊酯、丙酸戊酯、丁酸异丁酯、丁酸戊酯、丁酸乙酯、邻苯二甲酸二异丁酯等物质成分,而这些物质成分正好是果香味剂中进入到乳腺培养液中的风味物质,且当添加剂量为15μg/ml时,各物质成分含量为最高(P<0.05),之后呈下降趋势。果香味剂能显著影响奶牛乳腺上皮培养液中乳成分含量和风味。     

Identification of tumour initiating cells in feline head and neck squamous cell carcinoma and evidence for gefitinib induced epithelial to mesenchymal transition

Feline oral squamous cell carcinoma is considered a highly invasive cancer that carries a high level of morbidity. Despite aggressive surgery, patients often succumb to disease, the tumour having inherent insensitivity to radiation and chemotherapy. In this study we sought to identify cells within the feline SCC1 line that have stem cell properties, including inherent resistance mechanisms. When feline cells were subjected to harsh growth conditions, they formed sphere colonies consistent with a stem cell phenotype. Utilising CD133, we were able to identify a small fraction of cells within the population that had enhanced sphere-forming ability, reduced sensitivity to radiation and conventional chemotherapy and demonstrated resistance to the EGFR-targeting drug, gefitinib. In addition, long-term culture of feline SSC1 cells in gefitinib caused a change in cell morphology and gene expression reminiscent of an epithelial to mesenchymal transition. Taken together, these results suggest that feline SCC may be driven by small subset of cancer stem cells.     

Measurement of Carbonic Anhydrase Isozyme VI (CA-VI) in Bovine Sera,Saliva, Milk and Tissues

Concentrations of bovine carbonic anhydrase isozyme VI (CA-IV) in bovine serum, saliva, normal milk, colostrum, submandibular gland, liver, and mammary gland were determined. CA-VI was purified from bovine saliva and an antibody to CA-VI was generated. The concentrations of CA-VI in the saliva (7.8 ± 7.9 μg/ml), serum (2.1± 5.7 ng/ml), milk (7.9 ± 12.1 ng/ml), submandibular gland (284.7 μg/g protein), liver (921.0 ± 180.7 ng/g protein) and mammary gland (399.6 ± 191.2 ng/g protein) were determined by ELISA. No seasonal change in CA-VI levels was observed in normal milk. The concentration of CA-VI in colostrum (day 1 post partum) was 119 ng/ml and decreased rapidly by 1 month following birth. Mammary gland contained much smaller amounts than the submandibular gland. CA-VI mRNA was detected in the liver and mammary gland of cow by RT-PCR. The ELISA used in this study proved to be a precise and sensitive method for determining CA-VI concentrations in saliva, serum, milk and tissue specimens from cows. The ELISA may enable the study of changes in CA-VI associated with hereditary or metabolic disorders of the salivary gland, mammary gland and liver using small samples of saliva, serum or milk.     

奶牛乳腺上皮细胞原代培养、纯化及鉴定技术研究进展

奶牛乳腺上皮细胞不仅具有合成和分泌乳汁的功能,而且在乳腺的先天免疫中扮演着重要角色,对泌乳机制、乳房炎发病机制的研究,以及药物筛选具有重要意义。原代培养的奶牛乳腺上皮细胞适宜建立细胞模型,可作为生理、病理、药理等方面研究的良好介质,解决体内试验周期长、成本高、个体差异大的难题。作者主要从奶牛乳腺上皮细胞原代培养的发展历程、培养技术、纯化技术及鉴定方法等方面的最新研究情况进行综述,以期为奶牛乳腺上皮细胞培养相关研究提供参考。     

Research Progress on Primary Culture,Purification and Identification Technique of Bovine Mammary Epithelial Cells

The bovine mammary epithelial cells not only have the function of synthesis and secretion of milk, but also play an important role in innate immunity system of the mammary gland, and there is great significance of them on studying the mechanism of lactation, mastitis pathogenesis and drug screening.Primary cultured bovine mammary gland epithelial cells are suitable for setting up cell model which can be used as the dielectric for physiological, pathological and pharmacological researching, avoiding the difficulties of in vivo test, such as the long cycle, the high cost, the individual difference, etc.The author summarized the latest researches of cell primary culture in vitro, cultivation technology, purification and identification method in order to provide reference for the studies of bovine mammary epithelial cells culture.     

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。     

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.     

Comparison between stearoyl‐CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows

Stearoyl‐CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis‐double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non‐invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein‐Friesian cows and used for analysis of SCD expression by real‐time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9‐desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non‐invasive alternative to mammary tissue samples obtained by biopsy.