Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.     

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.     

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 10⁵ cells/cm² (high‐density group) or 0.8 × 10⁴ cells/cm² (low‐density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high‐density group and with two DC pulses of 25 V/150 µm for 10 µs in the low‐density group. The fusion rate (37.5%) in the high‐density group was significantly (P < 0.005) lower than in the low‐density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high‐density and low‐density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.     

细胞因子对牛乳腺上皮细胞体外增殖作用评价

分离纯化了乳腺上皮细胞,对细胞进行角蛋白免疫组化鉴定后,比较不同细胞因子对乳腺上皮细胞生长的影响.结果显示,生长因子EGF或HGF对牛乳腺上皮细胞的增殖具有重要作用,17β-E2不能促进牛乳腺上皮细胞的增殖,但可与EGF协同促进牛乳腺上皮细胞的增殖,表明17β3-E2对EGF诱导的乳腺上皮细胞增殖具有重要作用.     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。     

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells

Bovine colostrogenesis is distinguished by the specific transfer of IgG₁ from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E₂) and progesterone (P₄) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta‐2‐Microglobulin (β2M). We hypothesized that steroid hormone treatments (E₂ and P₄) of bovine mammary epithelial cells in vitro would induce up‐regulation of IgG₁ transcytosis candidate gene mRNA expression suggesting involvement in IgG₁ transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up‐regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG₁ transcytosis in bovine mammary cells.     

哺乳动物乳腺发育调控机制的研究进展

乳腺是哺乳动物特有器官之一,其发育可分为胚胎期、青春期、妊娠期、哺乳期和退化期共5个阶段,发育过程主要受激素、生长因子及细胞因子等调控。乳腺发育主要在出生后,青春期经生长激素(growth hormone, GH)、雌激素(estrogen, E)和胰岛素样生长因子1(insulin-like growth factor 1, IGF1)等调控启动分支形态发生;妊娠期及哺乳期由孕激素(progesterone, P)和催乳素(prolactin, PRL)共同作用产生腺泡并分泌乳汁;退化期则由断奶启动退化过程,腺体重塑至妊娠前状态。乳腺功能主要是通过产生和分泌乳汁以维持初生幼畜正常发育,研究乳腺发育调控机制对畜牧生产具有重要意义。本文以小鼠为例介绍其乳腺发育过程及发育调控机制,并阐述研究乳腺发育及人工调控乳腺发育对畜牧生产的深刻意义,为后期进一步研究乳腺发育和初乳形成提供一定理论参考。     

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。     

金黄色葡萄球菌对体外培养的奶牛乳腺上皮细胞PDGF-BB mRNA及蛋白表达的影响

为研究金黄色葡萄球菌(S.aureus)对奶牛乳腺上皮细胞(BMEC)中PDGF-BB m RNA及蛋白表达的影响,本研究采用热灭活的不同浓度的S.aureus菌液(0、105、106、108cfu/m L)作用于BMEC,分别在不同时间点(6 h、12 h、24 h、48 h)利用荧光定量PCR和western blot方法检测PDGF-BB m RNA及其蛋白的相对表达量。结果显示,不同浓度菌液处理组的PDGF-BB m RNA相对表达量随着作用时间延长表达量升高(p0.05)。6 h处理组随着菌液浓度的升高,PDGF-BB m RNA的相对表达量呈升高趋势;12 h处理组随着菌液浓度的升高,PDGF-BB m RNA的相对表达量呈降低趋势;24 h和48 h处理组105cfu/m L菌液处理组PDGF-BB m RNA相对表达量最高(p0.05)。同时,各时间点不同浓度菌液处理组PDGF-BB蛋白的相对表达量和m RNA表达基本一致。本研究表明,热灭活的S.aureus能够促进BMEC PDGF-BB m RNA和蛋白的表达。     

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.     

奶牛乳腺上皮细胞的克隆与特性分析

用组织块接种法分离培养奶牛乳腺上皮细胞,进行形态学观察、生长曲线的绘制和核型分析等生物学性状的检测,并进行酪蛋白基因的RT-PCR鉴定。结果显示,体外培养的奶牛乳腺上皮细胞生长旺盛,具有典型的上皮细胞形态特征;染色体数目为60;能够正常表达奶牛乳腺上皮细胞的2种特异性酪蛋白基因,即as1-酪蛋白基因和β-酪蛋白基因。表明成功获得了奶牛乳腺上皮细胞克隆。     

Moringa oleifera leaf flavonoids protect bovine mammary epithelial cells from hydrogen peroxide-induced oxidative stress in vitro

Bovine mammary epithelial cells (BMECs) of high-producing dairy cows are subject to constant oxidative stress as a result of high metabolic rate and physiological adaptation to intensive farming. Moringa (Moringa oleifera) leaf has been proposed to have the antioxidant potential in scavenging free radicals due to the presence of flavonoids. In this study, we investigated the cytoprotective effects of moringa leaf flavonoids in alleviating oxidative stress in BMECs in vitro. Oxidative stress was established by exposing isolated BMECs to H₂O₂ for 2 hr. Doses of moringa leaf flavonoids were evaluated by treating BMECs for 12 hr. The optimal concentrations of H₂O₂ and moringa leaf flavonoids were 500 μmol/L and 1.0 mg/ml, respectively. The results showed that moringa leaf flavonoids increased the activities of superoxide dismutase, catalase, and glutathione peroxidase; and reduced malondialdehyde activity and intracellular accumulation of reactive oxygen species (ROS) in the H₂O₂-induced oxidative stress system. A Hoechst33258 staining assay revealed that moringa leaf flavonoids decreased the apoptosis rate in BMECs, while leaving membrane integrity and nucleolar morphology unchanged. We concluded that moringa leaf flavonoids have the antioxidant capacity to effectively reduce oxidative stress in BMECs.     

不同供体细胞代数对兔体细胞核移植效率的影响

将兔成纤维细胞分为5～10低代数组和20～25高代数组进行核移植研究。结果显示,2试验组供体细胞核移植的效率存在差异。通过对供体细胞进行核移植后的重构胚胎的融合率、卵裂率、囊胚率以及体内发育的比较,发现低代数相对来说更有效率,但是高代数组也能获得正常的克隆后代。结果表明,兔不同代数体外培养细胞对克隆效率存在影响,并为制备转基因打靶兔提供基础。     

Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

Three‐dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long‐term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three‐dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up‐regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ‐casein could be detectable in a passage range in 3D culture. Expression of αs2‐casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long‐term study of lactation mechanisms in vitro.     

核移植技术在建立乳腺生物反应器中的应用

乳腺生物反应器的开发为制药业带来了生机,但目前采用的动物基因转移的方法,由于其各自固有的局限性,未能使得乳腺生物反应器的研究取得长足的进步。而核移植技术的发展为乳腺生物反应器的开发注入了活力。因此,笔者综合近年来国内外的文献,提出了核移植中供体细胞的选择以及处理方法,同时也从分子水平阐述了乳腺的一些调控机制,为核移植技术在乳腺生物反应器中的应用奠定了基础。     

无乳链球菌体外感染奶牛乳腺上皮细胞

用分离到的无乳链球菌(Streptococcus agalactiae,GBS)及标准菌株侵袭原代奶牛乳腺上皮细胞建立体外感染细胞模型,并进行细胞凋亡检测。同时,采用qRT-PCR方法检测细胞受到侵袭后白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子(TNF-α)等细胞炎性因子mRNA的转录水平。结果显示,标准菌株组(WL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组(FL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组与其标准菌株组的凋亡率差异也极显著(P<0.01)。qRT-PCR结果分析显示,标准株组TNF-α、IL-6及IL-8 mRNA转录水平与对照组相比,差异极显著(P<0.01);而分离菌株组TNF-αmRNA转录水平与对照组相比差异显著(P<0.05),IL-6及IL-8 mRNA转录水平差异不显著(P>0.05)。结果表明,与标准菌株相比,无乳链球菌分离株对奶牛乳腺上皮细胞侵袭和损伤能力较低,乳腺细胞的凋亡率也较低,当奶牛乳腺上皮细胞受到细菌侵袭的时候,细胞中IL-6、IL-8及TNF-α等炎性因子mRNA的转录水平都显著性提高,但在相同浓度下GBS标准株诱导细胞炎性因子的表达量显著高于GBS分离株。本试验结果为以后预防和控制GBS引起的隐性乳房炎提供试验依据。