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.     

Comparative study of the plasma globulin level, CD21(-) B-cell counts and FOXP3 mRNA expression level in CD4(+) T-cells for different clinical stages of feline immunodeficiency virus infected cats

Feline immunodeficiency virus (FIV) infection leads to hypergammaglobulinemia through mechanisms that remain poorly understood. We investigated changes in plasma globulin level, B cells, and T cells with progression of the clinical stage of FIV-infected cats. We classified FIV-infected cats into the stage of Asymptomatic carrier (AC) and AIDS-related complex (ARC) based on the clinical symptoms, and measured the plasma globulin level, the CD4(+) T-cell counts, and analyzed surface markers of B cells. We investigated the relationship between the plasma globulin level and regulatory T cells (Tregs) using the Forkhead box P3 (FOXP3) mRNA expression level. In FIV-infected cats, the plasma globulin level and the surface immunoglobulin (sIg)(+) CD21(-) B-cell counts were increased, whereas the CD4(+) T-cell counts were decreased compared with specific-pathogen free (SPF) cats. The mRNA expression of Blimp-1 (master gene of plasma cells) was increased in peripheral blood, and the FOXP3 mRNA expression level was decreased in CD4(+) T-cells. These immunological changes were marked in the ARC stage. These data indicate that the decrease of Tregs and the increase of plasma cells lead to hypergammaglobulinemia.     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

Immunohistochemical detection of HER-2/neu expression in spontaneous feline mammary tumours

Mammary gland tumours (MGT) are the third most common tumours in the cat. At least 85% are malignant and metastasis is common. The HER‐2/neu protooncogene encodes a 185‐kDa transmembrane tyrosine receptor kinase protein. Approximately 25–30% of human MGT demonstrate HER‐2/neu protein overexpression in the malignant cells, and overexpression has been associated with an increased metastatic propensity and a decreased prognosis. No reports have been published, to date, investigating the expression of Her‐2/neu in cats or cats with spontaneous mammary tumours. Based on the increased percentage of malignant mammary cancers in cats, compared to that in dogs, and the correlation of an increased malignancy and a decreased prognosis with Her‐2 overexpression in human mammary cancer, we hypothesized that cats with spontaneous malignant mammary adenocarcinoma overexpress Her‐2/neu in the neoplastic mammary epithelial cells. Thirty cats with MGT were assayed for Her‐2/neu immunohistochemical expression. The median percentage of cells from feline MGT expressing Her‐2/neu by utilizing the Dako polyclonal and CB11 monoclonal antibodies was 85 and 92.5, respectively. Her‐2/neu expression intensity grades 2 and 3 consistent with the overexpression by utilizing the Dako polyclonal and CB11 monoclonal antibodies were observed in 90 and 76.7% of cats with MGT, respectively. The level of overexpression concordance across the two antibodies was 70%. The results from this study suggest that Her‐2/neu overexpression is common in cats with spontaneous MGT, and therefore appears to represent an excellent model for Her‐2/neu‐overexpressing human breast cancer.     

Growth hormone and lactogenic hormones can reduce the leptin mRNA expression in bovine mammary epithelial cells

Leptin mRNA is expressed in not only adipocytes but also mammary epithelial cells and leptin protein is present in milk. Although milk leptin is thought to influence metabolism or the immune system in neonates, there is little information about the regulation of leptin expression in mammary epithelial cells. We examined the effect of growth hormone (GH) and/or lactogenic hormone complex (DIP; dexamethasone, insulin and prolactin) on leptin mRNA expression in mammary epithelial cells. We used a bovine mammary epithelial cell (BMEC) clonal line, which was established from a 26-day pregnant Holstein heifer. We confirmed that the mRNA was expressed in BMECs and the expression was significantly reduced by GH and/or DIP, when the cells were cultured on both plastic plates and cell culture inserts at days 2 and 7 after stimulation with lactogenic hormones. GH and/or DIP significantly increased level of alpha-casein mRNA in BMECs after 7 days on the cell culture inserts, but no mRNA expression was detected at day 2. GH and DIP significantly stimulated the secretion of alpha-casein from BMEC on cell culture inserts at 3.5 and 7 days. However, neither alpha-casein mRNA expression nor secretion was observed in the BMECs cultured on plastic dishes, even in the presence of GH or/and DIP. These results indicate that GH and DIP can directly reduce leptin mRNA expression in both undifferentiated and functionally differentiated bovine mammary epithelial cell.     

蛋氨酸对体外培养奶牛乳腺上皮细胞泌乳能力的影响

研究蛋氨酸与乳蛋白合成及乳腺泌乳能力的关系。建立体外培养的奶牛乳腺上皮细胞模型,采用高效液相色谱法、实时荧光定量PCR、细胞活力分析仪、甘油三酯试剂盒检测不同浓度蛋氨酸(0、0.2、0.4、0.8、1.2、1.6mmol/L)对体外培养24h的奶牛乳腺上皮细胞泌乳的影响。结果表明,蛋氨酸在1.2mmol/L时对体外培养的奶牛乳腺上皮细胞增殖的促进作用最明显,乳糖分泌量最高,β-酪蛋白mRNA表达量最高且甘油三酯合成最多(P<0.01)。     

Study on Expression of SYK in Dairy Cow Mammary Gland

To investigate the relationship between the expression of SYK and dairy cow mammary gland development and lactation, the expression of SYK in lactating dairy cow mammary gland with high or low quality milk and dry period Holstein dairy cow mammary gland was detected by Western blotting and laser confocal microscope.The results showed that SYK expression in dry period mammary gland was significant higher than that in lactating mammary gland (P<0.05).There was no SYK differential expression detected between lactating mammary gland with high quality milk and low quality milk (P>0.05).SYK was mainly located in the cytoplasm of ductal epithelial cells in dry period mammary gland.In lactating mammary gland, SYK was existed in acinar epithelial cells.All these results revealed that SYK was a regulator in mammary epithelial cell proliferation and differentiation.It participated in mammary gland reconstitution in dry period.     

脾源性酪氨酸激酶基因在奶牛乳腺中的表达研究

为探讨脾源性酪氨酸激酶(spleen tyrosine kinase,SYK)的表达与奶牛乳腺发育和泌乳功能之间的关系,试验采用Western blotting和激光共聚焦显微技术对泌乳期高乳品质、低乳品质及干乳期的中国荷斯坦奶牛乳腺组织中SYK的表达含量和表达部位的变化进行研究。结果表明,干乳期奶牛乳腺组织中SYK的表达显著高于泌乳期奶牛乳腺组织(P<0.05),泌乳期高乳品质、低乳品质奶牛乳腺组织中SYK的表达差异不显著(P>0.05);在干乳期SYK主要在乳腺导管上皮细胞的胞质中表达,而在泌乳期SYK在腺泡上皮细胞中表达。结果提示SYK是乳腺上皮细胞增殖与分化的调节因子,主要参与干乳期乳腺组织的重建过程。     

β-防御素在荷斯坦奶牛正常和炎性乳腺上皮细胞中的差异表达

为探讨荷斯坦奶牛乳腺上皮细胞在正常和炎性2种情况下β-防御素（BNBD5）的表达量是否存在差异,本研究通过添加内毒素（LPS）建立了实验性乳房炎的乳腺上皮细胞模型,并采用实时荧光定量RT-PCR方法检测了乳腺上皮细胞中BNBD5mRNA表达水平的变化。结果显示,添加LPS后乳腺上皮细胞中炎性因子IL-6、IL-12和TNF-α的mRNA表达量与空白对照组相比显著增加（P〈0.01）,并且α-酪蛋白mRNA表达量显著降低（P〈0.01）,说明添加LPS诱发上皮细胞产生了一定的炎性反应;并且当添加LPS终质量浓度为300μg/L并培养48h之后BNBD5的表达量最高,与空白对照组存在极显著差异（P〈0.01）;推测BNBD5基因可能参与了由LPS诱发的奶牛乳房炎的防御机制。     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

低水平葡萄糖对奶牛乳腺上皮细胞促炎症因子表达的影响

本研究旨在探讨低葡萄糖水平对奶牛乳腺上皮细胞中肿瘤坏死因子-α(TNF-α)、溶菌酶(LYZ)、诱导型一氧化氮合酶(iNOS)、白细胞介素-6 (IL-6)、乳铁蛋白(LF)和白细胞介素-8(CXCL8)等促炎症因子mRNA表达的影响。采用酶消化法将奶牛乳腺上皮细胞分离纯化后,分别在含有0.25、1.00和4.50 mg/mL葡萄糖的完全培养液中培养24 h,采用实时荧光定量PCR技术检测细胞中相关基因的表达。结果表明,上述基因的mRNA相对表达量在各葡萄糖浓度之间没有明显差异(P>0.05)。其中TNF-α和LYZ在各浓度葡萄糖中的表达量均非常少,iNOS的表达量居中,而IL-6、LF和CXCL8在各浓度组中的表达量相对较高。试验结果提示,葡萄糖浓度的减少并没有直接影响乳腺上皮细胞中TNF-α、LYZ、iNOS、IL-6、LF和CXCL8 mRNA的基础性表达;乳腺上皮细胞在未受到感染的情况下,能够表达一定数量的iNOS、IL-6、LF和CXCL8,这些因子可以作为先天性免疫防御的储备,在受到感染的情况下能迅速应对病菌的入侵。     

脂多糖诱导奶牛乳腺上皮细胞先天性免疫反应

采取荷斯坦奶牛乳腺,进行体外分离培养,并纯化细胞。用不同质量浓度(0、1、10、100mg/L)的脂多糖刺激乳腺上皮细胞,采用MTT法检测脂多糖对细胞增殖的影响,半定量PCR检测10mg/L的LPS对乳腺上皮细胞TLR4、TLR2、CD14、MD-2四个基因在不同时间(0、2、6h)mRNA表达水平的差异。结果表明,高剂量(100mg/L)的LPS对乳腺上皮细胞的增殖产生明显影响;LPS刺激乳腺上皮细胞后,导致TLR4、CD14、MD-2mRNA表达迅速升高,而TLR2mRNA弱表达。说明TLR4、CD14、MD-2参与LPS的识别,同时也说明脂多糖刺激乳腺上皮细胞后,乳腺上皮细胞能够产生先天性免疫反应。     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

Interleukin-8 expression by mammary gland endothelial and epithelial cells following experimental mastitis infection with E. coli

Epithelial and endothelial cells play a pivotal role in initiating and controlling the movement of leukocytes into tissues during inflammation through the production of cytokines and chemokines such as interleukin-8 (IL-8). In situ hybridization with an IL-8 riboprobe was used to determine IL-8 mRNA expression by mammary gland epithelial and endothelial cells in cows with experimental Escherichia coli mastitis. Epithelial cells of the gland, especially surrounding the alveoli, had increased IL-8 mRNA levels at all time points at which tissue samples were collected (8, 12, and 24h) after E. coli challenge. Levels of IL-8 expression in the epithelial cells decreased at 24h post-infection. IL-8 expression by mammary gland endothelial cells was low, but did increase slightly at 24h post-infection. Both epithelial and endothelial cells of the mammary gland can contribute to the production of IL-8 that is typically seen in coliform mastitis.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.     

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。