以奶牛乳腺上皮细胞(Dairy cow mammary epithelial cells,DCMECs)为研究模型,探讨microRNA-148b对DCMECs增殖和β-酪蛋白合成的影响.应用脂质体转染技术将miR-148b转染DCMECs,qRT-PCR观测其表达量,采用双荧光素酶实验验证miR-148b与转化生长因子B2(TGFB2)基因的靶向关系,分别应用EdU实验、酶联免疫吸附实验检测DCMECs的增殖以及β-酪蛋白含量.双荧光素酶实验结果证明TGFB2为miR-148b的靶基因,EdU实验结果显示,miR-148b可以促进DCMECs的增殖,ELISA结果表明过表达miR-148b可以促进β-酪蛋白合成.

利用从青春期奶牛乳腺中提取的类器官,在Ⅰ型胶原中以镶嵌式进行三维培养,用碱性成纤维细胞生长因子(basic fibroblast growth factor,FGF2)处理来建立奶牛乳腺分支模型.结果表明,在Ⅰ型胶原中以镶嵌式培养的青春期奶牛乳腺类器官,在FGF2处理下36 h时镜下即可见分支形态出现;且从36 h开始,FGF2组分支率显著高于对照组,表明奶牛乳腺分支化培养模型成功建立.结论:FGF2能使镶嵌在Ⅰ型胶原中的青春期奶牛乳腺类器官产生分支形态.

为探索miR-200b对奶牛乳腺上皮细胞泌乳能力的影响,以奶牛乳腺上皮细胞为体外研究模型,利用miR-200b mimics转染进行miR-200b过表达;应用生物信息学软件分析预测miR-200b靶基因,qRT-PCR检测miR-200b过表达后预测靶基因及乳成分合成相关信号通路关键基因mRNA表达的变化;通过CASY细胞活力分析仪及5-乙炔基-2′脱氧尿嘧啶核苷(EdU)标记法分别检测miR-200b过表达对细胞活力及增殖的影响;应用CSN2(β-酪蛋白)试剂盒、TG试剂盒及乳糖试剂盒检测细胞胞外分泌 β-酪蛋白、甘油三酯及乳糖含量的变化.生物信息学分析显示,miR-200b与乳腺泌乳功能基因Pten、Dnmt3a、Dnmt3b的3′UTR区序列具有互补结合的位点;qRT-PCR分析结果显示,与对照组相比,过表达miR-200b后的奶牛乳腺上皮细胞中Pten mRNA的表达量显著降低,而乳成分合成相关信号通路关键基因Akt、Srebp1、Csn2、Glut1 mRNA的表达量显著上调;miR-200b过表达能够提高奶牛乳腺上皮细胞活力,促进奶牛乳腺上皮细胞增殖及 β-酪蛋白、甘油三酯和乳糖的合成分泌.综上,miR-200b能够负调控靶基因Pten的表达进而在奶牛乳腺泌乳过程中发挥重要的正调控作用.

Peroxisome proliferator-activated receptor gamma (PPARγ) participates in lipogenesis in rats, goats, and humans. However, the exact mechanism of PPARγ regulation on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) remains largely unexplored. The aim of this study was to investigate the role of PPARγ regarding milk fat synthesis in DCMECs and to ascertain whether milk fat precursor acetic acid and palmitic acid could interact with PPARγ signaling to regulate milk fat synthesis. For this study, we examined the effects of PPARγ overexpression and gene silencing on cell growth, triacylglycerol synthesis, and the messenger RNA (mRNA) and protein expression levels of genes involved in milk fat synthesis in DCMECs. In addition, we investigated the influences of acetic acid and palmitic acid on the mRNA and protein levels of milk lipogenic genes and triacylglycerol synthesis in DCMECs transfected with PPARγ small interfering RNA (siRNA) and PPARγ expression vector. The results showed that when PPARγ was silenced, cell viability, proliferation, and triacylglycerol secretion were obviously reduced. Gene silencing of PPARγ significantly downregulated the expression levels of milk fat synthesis-related genes in DCMECs. PPARγ overexpression improved cell viability, proliferation, and triacylglycerol secretion. The expression levels of milk lipogenic genes were significantly increased when PPARγ was overexpressed. Acetic acid and palmitic acid could markedly improve triacylglycerol synthesis and upregulate the expression levels of PPARγ and other lipogenic genes in DCMECs. These results suggest that PPARγ is a positive regulator of milk fat synthesis in DCMECs and that acetic acid and palmitic acid could partly regulate milk fat synthesis in DCMECs via PPARγ signaling.

Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis.

Mammary gland development is controlled by several genes. Although miRNAs have been implicated in mammary gland function, the mechanism by which miR-486 regulates mammary gland development and lactation remains unclear. We investigated miR-486 expression in cow mammary gland using qRT-PCR and ISH and show that miR-486 expression was higher during the high-quality lactation period. We found that miR-486 targets phosphoinositide signaling in the cow mammary gland by directly downregulating PTEN gene expression and by altering the expression of downstream genes that are important for the function of the mammary gland, such as AKT, mTOR. We analyzed the effect of β-casein, lactose and triglyceride secretion in bovine mammary gland epithelial cells (BMECs) transfected by an inhibitor and by mimics of miR-486. Our results identify miR-486 as a downstream regulator of PTEN that is required for the development of the cow mammary gland.

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.

Milk is important for human nutrition, and enhanced milk quality has become a major selection criterion for the genetic improvement of livestock. Epigenetic modifications have been shown to be involved in mammary gland development; but the mechanisms underlying their effects remain unknown. MicroRNAs are involved in the regulation of milk synthesis and in mammary gland development. Our study is the first to investigate the roles of miR‐29s and epigenetic regulation in dairy cow mammary epithelial cells (DCMECs). Our results show that miR‐29s regulate the DNA methylation level by inversely targeting both DNMT3A and DNMT3B in DCMECs. The inhibition of miR‐29s caused global DNA hypermethylation and increased the methylation levels of the promoters of important lactation‐related genes, including casein alpha s1 (CSN1S1), E74‐like factor 5 (ElF5), peroxisome proliferator‐activated receptor gamma (PPARγ), sterol regulatory element binding protein‐1 (SREBP1), and glucose transporter 1 (GLUT1). The inhibition of miR‐29s reduced the secretion of lactoprotein, triglycerides (TG) and lactose by DCMECs. Moreover, the treatment of DCMECs with 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC) decreased the methylation levels of the miR‐29b promoter and increased the expression of miR‐29b. The link between miR‐29s and DNMT3A/3B enhances our understanding of the roles of miRNAs in mammary gland function, and our data will inform more experimentally oriented studies to identify new mechanisms of regulating lactation. We present new insights regarding the epigenetic regulation of lactation performance. Improved understanding of the molecular basis of lactation will aid in the development of strategies for optimizing milk quality in dairy cows and modifying the lactation performance of offspring. J. Cell. Physiol. 230: 2152–2163, 2015. © 2015 Wiley Periodicals, Inc.

The proteomics of inflammatory response in whey from cows with subclinical mastitis were analysed. Whey protein lysates were separated on 24 cm dry IPG strips (pH 3-10 linear) and 24 cm dry IPG strips (pH 4-7) using two-dimensional electrophoresis. The results indicated that the whey proteins in milk from cows with subclinical mastitis are different from those in milk from healthy cows. All protein spots were found to have biologically relevant changes in relative abundance during subclinical mastitis using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, including beta-1,4 galactosyltransferase, beta-2 microglobulin, complement 3, alpha-1-acid glycoprotein, beta-lactoglobulin A, alpha-S1 casein precursor, beta-casein B, and serotransferrin precursor. The mRNA expression of these genes was verified by quantitative real-time PCR. These proteins are involved in signal transduction, binding, transport, and immune defence activity. The results suggest that the markers may be used for the diagnosis of subclinical mastitis.

The role of LeuRS, an aminoacyl-tRNA synthetase, as an intracellular l-leucine sensor for the mTORC1 pathway has been the subject of much research recently. Despite this, the association between LeuRS and lactation in dairy cow mammary epithelial cells (DCMECs) remains unknown. In this study, we found that LeuRS expression in mammary gland tissue was significantly higher during lactation than pregnancy. Moreover, our data demonstrates that LeuRS is localized in the cytoplasm. Treatment with leucine increased DCMECs viability and proliferation, as well as mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase 1 (S6K1), p-S6K1, β-Casein, sterol regulatory element binding protein 1c (SREBP-1c), glucose transporter 1 (GLUT1), and Cyclin D1 mRNA and protein expression. Secretion of lactose and triglyceride were also increased. siRNA-mediated knockdown of LeuRS led to reduction in all of these processes. Based on these data, LeuRS up-regulates the mTOR pathway to promote proliferation and lactation of DCMECs in response to changes in the intracellular leucine concentration.

This experiment was to explore the influences of 5-aza 2′-deoxycytidine(5-Aza-dC)on the methylation status of promoter region of PPARγgene and its expression in dairy cow mammary epithelial cells(DCMECs).DCMECs were cultured invitro and treated with 0.1,0.5,1.0,5.0μmol/L 5-Aza-dC,methyltransferase activity was measured by EpiQuikTM DNA methyltransferase assay kit;CASY○R-technology was applied to measure cell viability and proliferation;bisulphite genomic sequencing PCR(BSP)technique was applied to detect the methylation state of the promoter region of PPARγgene in DCMECs;qRT-PCR was applied to detect PPARγmRNA expression;the protein expression level of PPARγwas determined by Western blot.Results showed that 0.1μmol/L 5-Aza-dC had no significante effect on cell viability and proliferation of DCMECs,and methyltransferase activity was significantly decreased at 9 6 h;compared with control group,methylation level of promoter region of PPARγgene in DCMECs was partially reduced after treating with 5-Aza-dC,and the mRNA and protein expression of PPARγwere increased significantly.Our results suggests that 5-Aza-dC can reduce methylation level of PPARγpromoter region,and promote the mRNA and protein expre s sion of PPARγgene in DCMECs.

为确定microRNA-152 (miR-152)对奶牛乳腺上皮细胞中乳蛋白合成的影响,本试验应用脂质体转染技术,改变miR-152在奶牛乳腺上皮细胞的表达量.采用实时荧光定量PCR、Western blotting、细胞活力分析等技术探索miR-152对奶牛乳腺上皮细胞增殖及泌乳功能的影响.结果显示,miR-152沉默,细胞因子信号传导抑制蛋白3(suppressor of cytokine signaling 3,SOCS3)表达增强,信号转导通路分子磷酸化信号转导与转录激活因子5(phospho-signal transducer and activator oftranscription 5,p-STAT5)、磷酸化哺乳动物雷帕霉素蛋白(phospho mammalian target of rapamycin,p-mTOR)、核糖体S6激酶1 (ribosomal protein S6 kinase 1,S6K1)、细胞周期蛋白D1(cyclinD1)表达减弱,细胞增殖能力减弱,β-酪蛋白分泌减少.研究结果表明,miR-152在奶牛乳腺上皮细胞调控的靶基因是SOCS3,通过抑制其表达而发挥作用.miR-152可通过调控p-STAT5、p-mTOR、S6K1、cyclinD1信号转导而促进乳蛋白的合成和乳腺上皮细胞增殖.

MicroRNAs (miRNAs) are a class of small non-coding, endogenous regulatory RNAs that function by controlling gene expression at the post-transcriptional level. Using small RNA sequencing and qRT-PCR techniques, we found that the expression of miR-152 was significantly increased during lactation in the mammary glands of dairy cows producing high quality milk compared with that in cows producing low quality milk. Furthermore, DNA methyltransferase 1 (DNMT1), which is a target of miR-152, was inversely correlated with the expression levels of miR-152 in the mammary glands of dairy cows. Dairy cow mammary epithelial cells (DCMECs) were used as in vitro cell models to study the function of miR-152. The forced expression of miR-152 in DCMECs resulted in a marked reduction of DNMT1 at both mRNA and protein levels. This in turn led to a decrease in global DNA methylation and increased the expression of two lactation-related genes, serine/threonine protein kinase Akt (Akt) and peroxisome proliferator-activated receptor gamma (Pparγ). In contrast, inhibition of miR-152 showed the opposite results. By using an electronic Coulter counter (CASY-TT) and flow cytometer, we discovered that miR-152 enhanced the viability and multiplication capacity of DCMECs. In conclusion, miR-152 plays an important role in the development and lactation processes in the mammary glands of dairy cows. Our data provide insights into dairy cow mammary gland development and lactation.

The protein expression and localization of leptin and its long form receptor(OB-Rb) were detected by western blottig methord and a confocal laser scanning microscope.The results showed that leptin protein level was higher in virgin,the lowest in lactation.Then OB-Rb was higher in virgin and pregnancy,lower in lactation,and recovered to the original level in involution.

Graphic organizers is a branch of the organizers,it is a practical tool for visual thinking.In combination with the language characteristics and requirements of the teaching of reading to explore the use of the KWL table,Author tool and Fishbone tool to help students read independently co-ordinate the process,deepen learning and clarity of thought rhetoric theory and methods.

In order to study the mRNA expression changes of VEGF-A,VEGF-C,and PLGF gene in different developmental periods of dairy cow mammol/lary gland,then lay the foundation for further research on the gene function of the cow mammol/lary gland.The SYBR Green Ⅰ dye Real-time PCR method was used,and the results were compared,with gene chip differential screening results of Affymetrix company.The results showed that three genes were expressed in 11 periods of mammol/lary gland.VEGF-A gene expression during late pregnancy and lactation was higher and significantly different from other periods(P0.05);VEGF-C gene expression was higher during lactation;PLGF gene expression from the onset of virgin to pregnancy increases gradually to 4 months pregnancy the highest expression level,followed by gradually decreasing the expression.The results could be drawn from the above,VEGF-A and VEGF-C gene may play a role in the lactating cows,and PLGF gene might be play an important role to cow mammol/lary gland development,so get the PLGF CDS sequence by PCR amplified method,lay the test foundation for further research on the gene function of PLGF gene.

Bovine whey contains many kinds of high-abundance proteins and varied interferential impurity,so it’s hard to obtain satisfied protein images of bovine whey in two dimensional electrophoresis(2-DE).Protein extraction from bovine whey is a key step to achieve high-resolution protein separation in 2-DE.Four routine total protein extraction methods were compared in order to determine an optimal one in 2-DE analysis for bovine whey.Bovine whey protein were extracted by direct schizolysis,TCA-acetone,trizol precipitation and 2-D clean up kit.The concentration of total protein was measured by 2-D quant kit,bovine whey protein were separated using SDS-PAGE and 2-DE.The data showed that use of 2-D clean up kit gave maximum protein yield,and the use of trizol gave the lowest.The result of SDS-