中药王不留行对奶牛乳腺细胞增殖及泌乳的影响

为确定中药王不留行对奶牛乳腺细胞泌乳功能的影响,以王不留行为原料,通过水浸提得到王不留行提取液,测出王不留行提取液中增乳活性物质邻苯二甲酸二丁酯含量,设置不同浓度中药提取液进行细胞试验,观察其对细胞泌乳的影响,筛选出增乳效力最显著的药物浓度。结果2 mg/mL(生药浓度)药物组增乳效果最显著,提示中药王不留行可直接作用于奶牛乳腺上皮细胞促进泌乳。     

王不留行增乳活性单体对奶牛乳腺上皮细胞增殖及泌乳性能的影响

应用CASY细胞分析仪及HPLC分别检测王不留行邻苯二甲酸二丁酯对乳腺上皮细胞增殖、细胞活力及乳腺上皮细胞分泌β-酪蛋白、乳糖的影响,研究王不留行增乳活性单体邻苯二甲酸二丁酯对奶牛泌乳中期乳腺上皮细胞增殖和泌乳性能的影响。试验结果表明,王不留行增乳活性单体成份邻苯二甲酸二丁酯对奶牛乳腺上皮细胞的增殖和细胞活力提高均显著(P0.05);能显著提高乳腺上皮细胞β-酪蛋白的表达(P0.05),也能提高乳糖的分泌(P0.05,P0.05)。因此,王不留行增乳活性单体邻苯二甲酸二丁酯可显著提高乳腺上皮细胞的增殖和泌乳能力。     

胰岛素、催乳素和氢化可的松对奶牛乳腺上皮细胞增殖和凋亡的影响

本研究旨在探讨胰岛素(A)、催乳素(B)和氢化可的松(C)三种泌乳激素对奶牛乳腺上皮细胞增殖和凋亡的影响。将正常的荷斯坦奶牛乳腺上皮细胞进行体外培养,采用L9(34)正交试验设计,以无血清无激素培养基作为对照,研究了在培养液中添加不同浓度的三种泌乳激素组合对奶牛乳腺上皮细胞增殖和凋亡的影响。结果表明,泌乳激素的添加能够促进奶牛乳腺上皮细胞的增殖,48 h之前,A2B3C1组合最有利于奶牛乳腺上皮细胞的增殖,60 h之后最优组合为A3B2C1即50 ng/mL催乳素、500 ng/mL胰岛素和0.1 ng/mL氢化可的松,三种激素对试验结果影响的主次顺序为A>C>B;泌乳激素对奶牛乳腺上皮细胞凋亡的影响不大,但A1B2C2,即500 ng/mL催乳素、50 ng/mL胰岛素和0.1 ng/mL氢化可的松组能减少凋亡细胞数量。说明,在正常的奶牛乳腺中,泌乳激素主要是通过促进奶牛乳腺上皮细胞的增殖为进一步的泌乳和乳品质的提高奠定坚实的生理基础。     

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。     

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。     

IGF-Ⅰ在奶牛乳腺的表达及其对乳腺上皮细胞泌乳功能的影响

为研究IGF-Ⅰ表达和奶牛乳腺发育与泌乳之间的关系,采用qRT-PCR检测奶牛乳腺组织中IGF-Ⅰ的表达情况,应用细胞培养、qRT-PCR、MTT法检测IGF-Ⅰ对奶牛乳腺上皮细胞的影响。结果显示,在青春期奶牛乳腺组织中IGF-Ⅰ的表达量最高;添加IGF-Ⅰ后,乳腺上皮细胞IGF-ⅠR表达增加,细胞增殖能力提高,β-酪蛋白表达增加。研究结果表明,IGF-Ⅰ可通过促进IGF-ⅠR的表达,从而促进乳腺上皮细胞增殖和提高乳中β-酪蛋白含量。     

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。     

王不留行对奶牛乳蛋白合成信号转导通路的影响

为确定王不留行对奶牛乳蛋白合成信号转导通路的影响,本试验通过水浸提方法制备王不留行提取液,采用Western blotting方法分别检测经王不留行水浸提液、雌激素及催乳素处理的泌乳期奶牛乳腺上皮细胞中乳蛋白合成信号分子的表达变化。结果显示,王不留行水浸提液、雌激素及催乳素都可以促进p-STAT5、p100、GAS、S6K1及p-mTOR的表达,进而促进乳蛋白合成,且添加蛋氨酸可以促进这一作用。提示,王不留行具有和雌激素、催乳素相似的作用,并在转录和翻译水平调节泌乳基因表达。     

奶牛乳铁蛋白肽真核表达载体的构建及其在奶牛乳腺上皮细胞中的表达

实验用奶牛乳铁蛋白肽特异性PCR引物从奶牛乳腺组织中扩增奶牛乳铁蛋白肽基因,然后将其连入pCMV-N-eGFP载体中,构建奶牛乳铁蛋白肽的真核表达载体GFP-LfcinB,并将其转染进奶牛乳腺上皮细胞中,分别运用蛋白质免疫印记和牛津杯法等方法检测GFP-LfcinB蛋白在细胞中的表达及其对金黄色葡萄球菌的抗菌活性.结果表明,试验成功的建立了奶牛乳铁蛋白肽的真核表达载体GFP-LfcinB,并且此重组载体能在奶牛乳腺上皮细胞中正常表达具有抗金黄色葡萄球菌活性的GFP-LfcinB蛋白.     

漏芦乙醇提取物对奶牛乳腺上皮细胞泌乳信号通路的影响

为了探讨漏芦乙醇提取物对奶牛乳腺上皮细胞泌乳信号通路的影响,试验以体外培养的奶牛乳腺上皮细胞为模型,先利用CASY细胞活力分析仪检测漏芦乙醇提取物(质量浓度分别为25,50,100,200,400μg/mL)作用于奶牛乳腺上皮细胞后乳腺上皮细胞的活力和增殖能力,选择添加对细胞生长增殖不受抑制即对细胞无毒性的漏芦乙醇提取物浓度来研究其对奶牛乳腺上皮细胞泌乳信号通路信号分子表达的影响。采用确定浓度的漏芦乙醇提取物处理细胞,然后用实时荧光定量PCR(qRT-PCR)检测奶牛乳腺上皮细胞泌乳信号通路关键信号分子信号转导与转录激活因子5(STAT5)、哺乳动物雷帕霉素靶蛋白(mTOR)、胆固醇调节元件结合蛋白1(SREBP1)、过氧化物酶增殖体激活受体γ(PPARγ)、丝氨酸-苏氨酸蛋白激酶1(AKT1)和葡萄糖转运蛋白1(GLUT1)基因的mRNA表达水平,以及采用Western-blot技术检测奶牛乳腺上皮细胞泌乳信号通路关键信号分子的蛋白质表达水平。结果表明:较高剂量(200,400μg/mL)的漏芦乙醇提取物显著抑制了奶牛乳腺上皮细胞活力及增殖能力(P0.05),因此选择25,50,100μg/mL的漏芦乙醇提取物来研究其对乳腺上皮细胞泌乳信号通路信号分子表达的影响;25,50,100μg/mL的漏芦乙醇提取物可显著提高细胞泌乳信号通路信号分子STAT5、mTOR、SREBP1、AKT1、GLUT1基因的mRNA表达水平(P0.05)及p-STAT5、p-mTOR、SREBP1、p-AKT1、GLUT1蛋白质表达水平(P0.05),但对PPARγ基因的mRNA表达及蛋白质的表达无显著影响(P0.05)。说明漏芦乙醇提取物能通过调节泌乳信号通路关键信号分子的表达,进而促进奶牛乳腺上皮细胞乳蛋白、乳脂和乳糖的合成。     

Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine mammary gland

Mammary gland growth and morphogenesis are regulated by interactions between hormones as much as by their individual actions. The effect of these interactions on the mammary gland phenotype in species other than rodents is relatively undefined. We investigated the individual and combined effects of estrogen (E), progestin (P), and prolactin (PRL) on mammary gland development in gilts. Pigs were shown to have a ductal-lobular parenchyma that underwent hormone-stimulated progression of terminal ductal lobular unit (TDLU) morphogenesis similar to that in the human breast. Ovariectomy plus hypoprolactinemia abolished mammary gland growth. Estrogen alone stimulated mammary epithelial cell proliferation, terminal bud formation, and the progression of TDLU1 structures to a TDLU2 morphotype. Maximal epithelial cell proliferation, DNA content, parenchymal area, and morphological development of the porcine mammary gland were realized following treatment with E + PRL or E + P + PRL. In contrast, P alone did not promote epithelial cell proliferation, TDLU type progression, mammary gland growth, or morphogenesis. These data indicate that interactions between E and PRL are the main determinants of growth and morphogenesis in the porcine mammary gland.     

四种植物提取物对大肠杆菌的抑菌效果研究

 为充分利用当地资源和进一步开发植物药进行畜禽大肠杆菌病防治提供科学依据,采摘了本地生长的马鞭草、马齿苋、忍冬藤、蒲公英等四种植物,应用水提醇沉法制备提取液,用纸片扩散法和试管两倍稀释法对猪源、鸽源、牛源大肠杆菌进行体外抑菌效果试验,同时以小鼠为试验动物,检测四种植物提取液体内的抗菌作用。结果显示,四种植物对猪源大肠杆菌的抑菌圈在10～14 mm之间,均为中敏;对鸽源大肠杆菌在10～22 mm之间,马鞭草极敏,蒲公英高敏;对牛源大肠杆菌在7～14 mm之间,马鞭草高敏,蒲公英、忍冬藤中敏,马齿苋低敏。马鞭草对猪源、鸽源大肠杆菌的MIC和MBC是31.25 mg/mL,对牛源是62.5 mg/mL;蒲公英对猪源、鸽源的大肠杆菌MIC和MBC是62.5 mg/mL,对牛源是125 mg/mL;马齿苋对猪源的MIC和MBC是62.5 mg/mL和125 mg/mL,对鸽源、牛源的MIC和MBC分别是125 mg/mL和250 mg/mL;忍冬藤对猪源、牛源的MIC和MBC分别是125 mg/mL和250 mg/mL,对鸽源MIC和MBC是62.5 mg/mL和125 mg/mL。小鼠体内抗菌作用提示,用蒲公英和马鞭草1.88 g/kg、马齿苋和忍冬藤3.75 g/kg进行预防,可使感染大肠杆菌的小鼠成活率为达100%。四种植物提取液对大肠杆菌体外抑菌和体内抗菌均有较好的效果,马鞭草效果最好     

New developments on the galactopoietic role of prolactin in dairy ruminants

In most mammals, prolactin (PRL) is essential for maintaining lactation and its suppression strongly inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear because inconsistent effects on milk yield have been observed with short-term suppression of PRL by bromocriptine. By contrast, in vitro studies have provided evidence that PRL helps to maintain the differentiation state and act as a survival factor for mammary epithelial cells. Therefore, a series of experiments were conducted to assess the galactopoietic role of PRL. In a first experiment, daily injections of the PRL inhibitor quinagolide reduced milking-induced PRL release and induced a faster decline in milk production. Milk production was correlated with PRL released at milking. Quinagolide reduced mammary cell activity, survival, and proliferation. During the last week of treatments, differential milking (1× vs 2×) was applied. The inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the udder half milked 1×, suggesting that the response to PRL is modulated at the gland level. In a second experiment, cows were injected with quinagolide, quinagolide + injection of bovine PRL at milking time, or water. As in the first experiment, quinagolide reduced milk, protein, and lactose yields. Although PRL injections at milking time were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of milk-purified mammary epithelial cells. Recently, we investigated the use of quinagolide at drying off. Treating late-lactation cows with quinagolide decreased milk production within the first day of treatment and induced faster increases in somatic cells and bovine serum albumin content in mammary secretions after drying off, which indicates an acceleration of mammary gland involution. In conclusion, these data, combined with data from other studies, provide a good body of evidence indicating that PRL is galactopoietic in dairy cows. However, the response to PRL appears to be modulated at the mammary gland level.