大鼠不同时期乳腺组织中雌激素孕激素及催乳素的变化规律

为探讨雌激素(E2)、孕激素(P)及催乳素(Prl)在大鼠不同时期乳腺组织中的变化规律,选雌性SD大鼠42只,分别为处女鼠、妊娠鼠(6d、12d、18 d)、泌乳鼠(6d、12d、18 d),每个时期6只.在指定的时间点处死大鼠取乳腺组织.用放免(RIA)组织匀浆中E2、P及Prl水平进行定量研究,用统计学方法进行处理,观察这三种激素的变化规律.结果显示E2从处女期到整个妊娠期,其呈现下降趋势.而分娩后E2水平急剧升高,后又有所下降,但维持在较高水平;在乳腺发育不同时期,P在乳腺组织中整体水平一直呈现下降趋势;Prl水平以处女期最高;随着妊娠进行逐渐降低,整个乳腺发育过程中Prl整体上几乎一直呈下降趋势.表明E2、P及Prl在促进乳腺发育及维持和启动泌乳方面发挥了重要的作用.     

大鼠不同时期血液中雌激素及孕激素的变化规律研究

为探讨大鼠血液中雌激素、孕激素的变化规律,试验选择雌性SD大鼠42只,分别为处女鼠(0～90 d,第90 d开始交配)、妊娠鼠(96 d、102 d、108 d)、泌乳鼠(114 d、120 d、126d),每个时期6只.在指定时间点处死大鼠取血液,用放射免疫法(RIA)对雌激素、孕激素进行定量研究,观察雌激素、孕激素...     

催乳素及其受体对乳腺发育研究进展

本文主要对催乳素受体的结构、作用机理、功能及其在不同时期的乳腺发育的不同作用进行的论述。总结了近几年来的有关研究进展,为进一步的研究工作提供参考资料。     

经产母兔血液中雌二醇与催乳素的变化规律

本试验主要开展不同的诱导经产哺乳期母兔同期发情方法对母兔血液循环中雌二醇和催乳素的影响。48只经产母兔随机分为4组,每只经产母兔在哺乳期哺乳8只以上的仔兔。试验设计:试验Ⅰ组在母兔分娩后2天时,采用48h母仔隔离方式;试验Ⅱ组在母兔分娩后2天时,注射25IU PMSG;试验Ⅲ组在母兔分娩后的第3天进行24h的母仔隔离处理;试验Ⅳ组为对照组。所有试验母兔均在分娩后的第4天进行人工授精,在人工授精后立即注射1μg的促排卵激素。试验动物在分娩后的第2、3、4天的早上9:00采集血液。雌二醇采用免疫电泳法测定,催乳素用免疫放射法测定。所有的试验母兔的血液雌二醇在分娩后的第2天和第3天有相似的水平(第2天:113.24±13.7pg/mL;第3天:142.01±13.7pg/mL)。在分娩后的第4天,注射了PMSG试验兔、母仔隔离24h试验兔、母仔隔离48h试验兔的血液雌二醇水平比分娩后第3天分别高出63.07%、73.12%、93.47%(P<0.001)。而且,母仔隔离48h试验兔在人工授精时,血液雌二醇水平比对照组高,差异极显著(267.42±27.84 pg/mL;172.33±26.76 pg/mL;P<0.001)。在各试验组之间,同一时间点的血液催乳素水平之间是无差异的。Ⅰ、Ⅱ、Ⅲ、对照组各组的受胎率分别为:91.67%、66.66%、83.33%、83.33%;初生窝产活仔数分别为:9.0±0.9、10.4±0.8、9.7±0.9、8.9±0.9;25日龄断奶仔兔数分别为:8.4±0.5、8.4±0.5、7.7±0.5、7.7±0.6;但是试验组Ⅱ的初生死胎数(1.12±0.3)要高于其他组(0.1±0.3,0.08±0.25,0.1±0.3)(P<0.05)。     

催乳素对奶牛乳腺上皮细胞酪蛋白合成的影响

试验研究催乳素对奶牛乳腺上皮细胞(BMECS)乳蛋白合成的影响.选用健康、状态良好的荷斯坦奶牛乳腺组织为试验材料,分离培养纯化BMECs,选取P3代BMECs进行试验.根据催乳素的添加浓度分为5组(0、100、200、400、800μg/L),催乳素处理24 h.结果 显示,对照组相比,添加100、200 μg/L催乳...     

催乳素刺激牛乳腺中脂肪生成基因的表达但不刺激葡萄糖转运蛋白的表达

在泌乳开始时,葡萄糖转运蛋白(GLUT)和参与乳脂合成的一些酶的表达呈显著增加,本试验旨在研究催乳素是否刺激这些基因的表达。试验分为:无激素(对照组)、胰岛素样生长因子Ⅰ组、胰岛素(Ins)组、胰岛素+氢化可的松+羊催乳素(InsHPrl)组、胰岛素+氢化可的松+催乳素+17β-雌二醇(InsHPrlE)组,牛乳腺组织培养采用激素处理48、72和96h。试验采用实时荧光定量PCR检测β-酪蛋白、α-乳清蛋白和甾醇调节元件结合因子1(SREBF1)、脂肪酸合成酶(FASN)、乙酰基辅酶A羧化酶(ACACA)、stearyol-CoA去饱和酶(SCD)、葡萄糖转运蛋白1、葡萄糖转运蛋白8和葡萄糖转运蛋白12的相对表达量。结果表明,催乳素混合组InsHPrl和InsHPrlE处理96h后,乳腺组织块中β-酪蛋白和α-乳清蛋白mRNA的表达增加了数百倍,同时也极显著提高了SREBF1、FASN、ACACA和SCD基因的表达(P<0.01)。然而,InsHPrl和InsHPrlE处理72h后,GLUT1或GLUT8表达量无明显变化,而GLUT12表达量显著降低了50%(P<0.05)。单一催乳素刺激组的小鼠乳腺上皮细胞系HC11或牛原代培养乳腺上皮细胞中,GLUTs表达不增加。此外,在催乳素刺激的奶牛乳腺中,GLUTs表达也无明显变化。说明在泌乳开始时,催乳素明显的增加了乳蛋白和脂肪生成基因的表达,但并没有显著上调GLUT基因的表达。     

中药王不留行增乳活性单体及催乳素对奶牛乳腺上皮细胞特异性miRNA的影响

本试验旨在研究中药王不留行增乳活性单体邻苯二甲酸二丁酯及催乳素对奶牛泌乳中期乳腺上皮细胞miRNAs表达的影响;荧光定量RT-PCR检测增乳活性单体邻苯二甲酸二丁酯及催乳素作用后乳腺上皮细胞miRNAs表达变化;王不留行增乳活性单体邻苯二甲酸二丁酯及催乳素均抑制原代培养的泌乳中期奶牛乳腺上皮细胞miRNA-143、miRNA-125和miRNA-195表达;邻苯二甲酸二丁酯可抑制miRNA-21表达,催乳素对miRNA-21表达的影响尚不确定。首次阐明中药王不留行增乳活性单体邻苯二甲酸二丁酯和催乳素能引起乳腺上皮细胞miRNAs表达变化。     

胰岛素、催乳素对奶山羊乳腺上皮细胞泌乳功能的影响

具有泌乳功能的乳腺上皮细胞可作为乳腺生长发育及泌乳功能调控机制研究的细胞模型。本研究对已建立的奶山羊乳腺上皮细胞系的泌乳功能进行评价。应用SDS-PAGE、TG试剂盒及HPLC方法对体外培养奶山羊乳腺上皮细胞总蛋白、乳脂和乳糖的分泌情况进行了测定以确定胰岛素、催乳素处理后的奶山羊乳腺上皮细胞的泌乳功能。结果表明：在添加表皮生长因子、胰岛素样生长因子-1、转铁蛋白-硒钠、标准胎牛血清的DF12培养液中奶山羊乳腺上皮细胞具有乳脂、乳蛋白及乳糖的分泌能力。胰岛素处理48 h后对细胞的泌乳功能无显著影响,催乳素处理细胞48 h后可使奶山羊乳腺上皮细胞乳蛋白、乳糖分泌能力显著升高。从而建立了具有泌乳功能的奶山羊乳腺上皮细胞系,为奶山羊、奶牛等重要经济动物泌乳机制的研究提供了便利条件。     

泌乳周期中外源物质对大鼠乳腺内肥大细胞数量的影响及乳腺组织内组胺含量的研究

选择泌乳期和静止期大鼠，经乳腺局部（乳导管）或外周（跖部皮下）注射辣根过氧化物酶（HRP）后，应用改良甲苯胺蓝染色法（MTB）对乳腺及其引流（鼠蹊部）和非引流（腘）淋巴结内的肥大细胞数量变化进行了研究，用荧光测定改良法检测了乳腺组织中组胺含量。结果显示，对照组乳腺及引流和非引流淋巴结内的肥大细胞数量均为静止期显著多于泌乳期（P〈0．01），乳腺中组胺含量也呈相同的变化趋势。在泌乳期从不同部位引人HRP后，乳腺及乳腺引流淋巴结内肥大细胞数量明显增多，与对照组相比差异极显著（P〈0．01）；而乳腺非引流淋巴结内肥大细胞数量则略有减少，与对照组相比差异不显著（P〉0．05）。在静止期从不同部位引人HRP后，乳腺及乳腺非引流淋巴结内肥大细胞数均减少，而乳腺引流淋巴结内的肥大细胞数则随引人部位不同而有差别：乳腺引人时增加，而外周引入时减少。本研究结果表明肥大细胞数量随生理周期的改变而增减的趋势与乳腺的发育、乳腺局部免疫状态及内分泌调节都有关系，而且不同泌乳阶段乳腺内肥大细胞对异物的易感性也存在差异。     

胰岛素、催乳素和孕酮对奶牛乳腺上皮细胞泌乳功能的影响

具有泌乳功能的乳腺上皮细胞系可作为乳腺发育学、乳腺病理学、泌乳生物工程学研究的细胞模型,本研究测定了激素对奶牛乳腺上皮细胞系泌乳功能的影响,为阐述泌乳机制提供工作基础。应用HPLC方法对体外培养奶牛乳腺上皮细胞的酪蛋白和乳糖的分泌情况和细胞培养液中的酪蛋白、乳糖含量进行测定,以确定胰岛素、催乳素和孕酮处理后的奶牛乳腺上皮细胞的泌乳功能。试验结果表明：奶牛乳腺上皮细胞具有酪蛋白和乳糖的分泌能力,在72h内,随着细胞培养时间延长,胰岛素处理组细胞中的酪蛋白和乳糖的升高趋势不明显,孕酮处理组升高趋势较明显,催乳素处理组升高趋势很明显;胰岛素处理组、催乳素处理组和孕酮处理组细胞培养液中酪蛋白升高趋势均很明显,乳糖含量均很高,三组激素比较而言,胰岛素处理组乳糖含量最高。此外,三组激素对乳腺上皮细胞活力影响均很大,在72h之内,总体变化为：催乳素处理组和孕酮处理组细胞活力均升高,胰岛素处理组细胞活力下降。     

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers

MicroRNAs (miRNAs) are small non‐coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein‐Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty‐two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta‐miR‐10b, bta‐miR‐29b, bta‐miR‐101, bta‐miR‐375, bta‐miR‐2285t, bta‐miR‐146b, bta‐let7b, bta‐miR‐107, bta‐miR‐1434‐3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF‐beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.     

Effects of tripeptides and lactogenic hormones on oligopeptide transporter 2 in bovine mammary gland

This study was conducted to investigate the expression of oligopeptide transporter 2 (PepT2) and its potential function in bovine mammary gland. First, the PepT2 mRNA and protein were determined in cultured mammary epithelial cells. Then the effects of lactogenic hormones (prolactin, hydrocortisone or insulin) and substrate (threonyl-phenylalanyl-phenylalanine) on PepT2 were investigated. The PepT2 mRNA and protein were successfully detected in bovine mammary epithelial cells. PepT2 gene expression was enhanced by the addition of 50, 500 and 5000 ng/ml prolactin, 10 and 100 ng/ml hydrocortisone, and 50, 500, 5000 and 50,000 ng/ml insulin. PepT2 mRNA abundance was increased when 5, 10 and 15% of threonyl-phenylalanyl-phenylalanine was included. Responses of PepT2 to lactogenic hormones and oligopeptide inferred that it may play an important role in bovine mammary gland.     

Effects of steroid hormone treatment on mammary development in prepubertal heifers

The objective of this study was to determine the effects of steroid hormone implantation in heifer calves on the ability of mammary tissue to develop subsequently in organ culture. Twenty-four calves were paired by date of birth and assigned to groups (eight calves/group). At 4, 7, or 10 mo of age, calves were implanted subcutaneously (s.c.) with pellets containing cholesterol or cholesterol, 17β-estradiol, and progesterone for 9 or 18 d. The calves were euthanized and uteri and mammary glands were removed and weighed. Slices of mammary parenchymal tissue were incubated for 5 d at 37°C in a 50% O₂, 5% CO₂ humidified atmosphere in Waymouth’s 752/liter medium supplemented with insulin (5.0 μg/ml) or lactogenic hormone complex insulin (5.0 μg/ml), aldosterone (0.1 μg/ml), hydrocortisone (0.1 μg/ml), and prolactin (1.0 μg/ml) in the presence or absence of epidermal growth factor (EGF) (0.06 μg/ml) to promote lobulo-alveolar development. Tissue sections were stained and mounted on slides for morphologic and histologic analysis or prepared to evaluate expression of β-casein mRNA. There were no morphologic differences in slices from calf mammary tissues despite age, steroid hormone priming, or hormones used in tissue culture. The 4-mo-old calves required steroid priming followed by incubation of the tissue slices with the lactogenic complex with or without epidermal growth factor to induce cytological changes associated with lactogenesis but did not express β-casein mRNA. At 7 mo of age, steroid hormone priming was not necessary for induction of alveolar formation and secretion. Incubation of the tissue slices from 7-mo-old calves with the lactogenic complex was sufficient to induce alveolar formation and secretion. However, β-casein mRNA was not expressed. At 10 mo of age, exposure of tissue from calves to the lactogenic hormones caused histologic changes reminiscent of the ability to secrete milk regardless of hormone priming. However, estrogen and progesterone priming was necessary before incubation of the tissue slices with the lactogenic hormones to induce β-casein mRNA expression. When epidermal growth factor was added to the lactogenic hormone complex, β-casein mRNA expression decreased. These data support the concept that there is a sequential development of responsiveness of the mammary gland to various hormones. By 10 mo of age, prepubertal heifers reach a stage of maturity where steroid hormone priming followed by incubation of tissue slices with the lactogenic hormones is sufficient to induce both structural and functional differentiation.     

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。     

健康奶牛与临床型乳腺炎奶牛乳腺线粒体蛋白组差异表达分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构再进行蛋白质组学研究,可提高低丰度蛋白在双向凝胶电泳中的检出率。通过对比分析乳腺炎奶牛乳腺与正常奶牛乳腺线粒体蛋白质组的表达变化,为奶牛乳腺炎的生物学治疗及抗病育种工作筛选出目基因和蛋白。超速离心法分离线粒体,双向凝胶电泳分离蛋白,PDQuest7．4软件分析差异蛋白斑点,高效液相色谱串联离子阱质谱鉴定差异蛋白。从奶牛乳腺线粒体蛋白2-DE图谱中筛选出17个差异表达的蛋白质斑点,质谱鉴定出17个差异表达蛋白（6个蛋白在奶牛乳腺炎发生过程中下调,8个上调,1个只在正常情况下表达,2个只在乳腺炎乳腺组织中表达）。筛选出的差异蛋白质涉及到细胞的能量代谢、蛋白质合成、mRNA的加工成熟及调亡调控等许多方面,表明奶牛乳腺炎发生时乳腺线粒体组织结构和代谢状态都发生了明显的变化。     

人血清白蛋白载体的构建及其在山羊乳腺中的瞬时表达

根据GeneBank中人血清白蛋白(HSA)的基因序列,设计一对特异性的引物,从人肝cDNA文库中扩增出其编码序列,并加入酶切位点XhoⅠ,以pCDNA 3.1为模板,设计特异性的引物,并在上游引物加入NotⅠ,下游加入ApaⅠ和NotⅠ,扩增出筛选标记新霉素抗性基因neo,然后再以pBC1为基本骨架,在其NotⅠ、XhoⅠ位点处分别插入hsa和neo,从而得到乳腺表达载体pBC-NEO-HSA,将该质粒注射入奶山羊乳腺中进行暂时性的表达,结果显示所构建的载体能够有效地指导目的基因的表达,从而为下一步构建细胞系、克隆动物作好基础。     

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.     

Haemoglobin in normal and neoplastic canine mammary glands

Four types of globins for oxygen transport are known in vertebrates, and the haemoglobin is responsible for carrying oxygen in blood. In this study, we found that haemoglobin was also expressed in canine mammary glands. Samples were taken from 26 malignant mammary tumors, 16 normal mammary glands and 10 other normal tissues. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and mass spectrometry were used to investigate haemoglobin in mammary tissues. The results indicated that normal canine mammary glands expressed high levels of haemoglobin protein as shown by Coomassie blue staining. The identity of haemoglobin was confirmed by immunoblotting and mass spectrometry, and the mass spectrometry data revealed that both alpha-haemoglobin and beta-haemoglobin were expressed. Relative to normal mammary glands, the levels of haemoglobin expression in mammary tumors were lower. Our results also indicated that the haemoglobin was endogenously produced in mammary gland tissues and was not derived from the erythroid cells.     

Decorin concentrations in canine normal and neoplastic mammary gland tissues

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.