Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate (DBP) separated from Vaccaria segetalis on the expression of lactation signal transduetion genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγand elf5, will be helpful to reveal the molecular mechanism. Western blot and qRTPCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Colocalization expression of prolactin receptor (PRLR) and estrogen receptor α (ERα) was observed by immunofluoreseence;the expression changes of miRNAs (21, 125b, 143, and 195) and the secretion of β-casein and lactose were detected byqRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin (PRL) in dairy cow mammary gland epithelial cells (DCMECs), increased the expressions of prlr, erα, akt1,and elf5 genes, while repressed ppaγ expressions. DBP promoted socs2 mRNA expression, but its protein expressionswere repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA-195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain.DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.     

Proteomic Identification of Differentially Expressed Proteins in Vaccaria segetalis-treated Dairy Cow Mammary Epithelial Cells

To investigate the potential effects of Vaccaria segetalis, we employed the proteomic approach on the dairy cow mammary gland epithelial cells (DCMECs). A total of 35 differentially expressed nuclear proteins were visualized by 2-DE and silver nitrate staining. Of these, five proteins also displayed significant expression changes upon treatment of the water decoction from Vaccaria segetalis, and such alterations were further confirmed by RT-PCR. Together, at both the mRNA and protein levels, the water decoction from Vaccaria segetalis increased the expression of proteins ethylmalonic encephalopathy 1 (ETHE1), vesicle amine transport protein 1 homolog (VAT1), parkinson disease protein 7 homolog (Protein DJ-1), proteasome subunit alpha type-2 (PSMA2) and SUMO-activating enzyme subunit 1 (SAE1). This study would enable a better understanding of the molecular mechanisms underlying this water decoction effects at the protein level.     

奶牛催乳素(PRL)及其受体(PRLR)基因研究进展

催乳素(prolactin)参与了很多生理活动,包括乳蛋白的合成、免疫活动的调节、促进生殖器官的发育、维持渗透压的平衡以及一些生理行为.催乳素要行使其生物学功能必须与其受体(prolactin receptor)结合.本文就PRL基因及其受体PRLR基因的结构、功能以及在奶牛上的研究进展作一综述.     

Galactopoietic Activity of Dibutyl Phthalate Isolated from Vaccaria segetalis

A galactopoietic compound, identified as dibutyl phthalate（DBP）, was isolated from Vaccaria segetalis. The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells（DCMECs） cultured in vitro and dairy cow was evaluated. Results showed that DBP could promote cell viability, proliferation ability, lactose and β-casein secretion of DCMECs, which could also raise the milk yields of dairy cows significantly.     

FLCN对奶牛乳腺上皮细胞增殖及泌乳功能的影响

FLCN基因参与多种代谢途径和细胞过程,国内外关于FLCN在奶牛乳腺发育过程中表达及调节的研究鲜有报道。应用RNA干扰技术和质粒转染技术改变FLCN基因在奶牛乳腺上皮细胞中表达量,流式细胞仪检测细胞增殖,采用qRT-PCR和Western blot检测FLCN对泌乳相关功能基因AMPK、mTOR、CyclinD1、Caspase3和β-酪蛋白表达的影响。结果表明,FLCN正向调节mTOR磷酸化水平,促进乳蛋白合成和细胞增殖,抑制细胞凋亡,负调控能量代谢调节子AMPK。FLCN在奶牛乳腺上皮细胞中可通过mTOR信号通路调控细胞增殖及乳蛋白合成,研究对揭示FLCN调控奶牛乳腺上皮细胞增殖和泌乳具有重要作用。     

泌乳奶山羊非繁殖季节诱发发情研究

奶山羊具有明显的季节性发情的特点,在非繁殖季节里,泌乳母羊体内的催乳素水平要高于繁殖季节,从而抑制了卵巢的生理机能。本研究采用具有抑制催乳素分泌作用的溴隐亭(CB-154,Parlodel)药物来降低母羊体内的催乳素水平,配合使用促性腺激素,提高泌乳母羊的诱发发情率,使配种后不返情率达到80％(28/35)。     

娟姗牛催乳素基因型与产奶量的关联性分析

[目的]研究娟姗牛催乳素（PRL）基因多态性与产奶量之间的关联性,为选育高产奶量娟姗牛新品系奠定基础.[方法]利用PCR-RFLP分析44头娟姗牛PRL基因外显子2和外显子4的多态性,统计不同基因型娟姗牛305 d的平均产奶量,然后分析娟姗牛PRL基因型与产奶量的关联性.[结果]娟姗牛PR基因外显子2和外显子4存在RsaI酶切多态性,外显子2有AA和AG两种基因型,外显子4有AA、AG和GG 3种基因型;外显子2和外显子4的AG型频率分别是0.7272和0.5454,为优势基因型.PRL基因两个外显子AA型个体的305 d平均产奶量均高于AG型和GG型（P＞0.05）;基因型组合效应分析发现,A2A2A4G4为优势组合基因型,但在305 d平均产奶量方面是A2G2A4A4组合基因型的产奶量显著高于A2A2A4A4、A2A2A4G4和A2A2G4G4组合基因型（P＜0.05）.[结论]娟姗牛PRL基因外显子2和外显子4的碱基突变与产奶量之间存在关联性,其中第8398位碱基A是娟珊牛高产奶量的有利等位基因.     

籽鹅催乳素基因的克隆及诱导表达条件的优化

运用RT-PCR方法,获得籽鹅脑垂体催乳素（Prolactin,PRL）基因编码区序列cDNA,克隆到pMD18-T载体上。DNA序列分析表明,PRL cDNA全长690 bp,编码230个氨基酸残基,与皖西白鹅的碱基同源性达99.57%,氨基酸同源性达99.56%。将所得序列与表达载体pET-32 a（＋）构建表达质...     

水牛 STAT5a 和 STAT5b 基因启动子克隆及其活性分析

为了解转录激活与信号转导因子5(STAT5)在水牛乳腺发育及泌乳生理中的功能,对水牛STAT5a和STAT5b基因的5′调控区启动子序列进行了克隆,并比较其活性.根据GenBank已公布的牛STAT5a和STAT5b基因序列设计特异性引物,以广西本地水牛乳腺组织基因组DNA为模板,通过PCR法分别扩增不同长度STAT5a和STAT5b基因启动子片段并进行生物信息学分析.结果表明:克隆得到的水牛STAT5a基因启动子片段(P1和P2)大小是500bp,700bp,STAT5b基因启动子片段(P3,P4和P5)大小是500bp,800bp,1 500bp.在线分析结果显示,仅P2片段中存在高甲基化位点,且富含SP1,AP2等转录因子结合位点.将构建的不同长度启动子片段分别连入pGL3-Basic载体,分别转染水牛乳腺上皮细胞,检测荧光素酶表达水平.与未转染组相比,P1~P5质粒转染组的荧光素酶比值均显著提高(p0.05);添加5mg/mL质量浓度催乳素(PRL)处理乳腺上皮细胞,P3质粒转染组的荧光素酶比值显著高于其他各组(p0.05).以上结果表明,水牛STAT5a和STAT5b基因启动子活性均受到PRL调节,两者表达水平在水牛乳腺上皮细胞中不同,且STAT5b基因表达受PRL调控更为明显.     

奶牛乳腺长链脂肪酸转运的研究进展

奶牛乳脂中的长链脂肪酸（LCFA）JL乎全部来源于从消化道吸收并转运人血液的食物外源性LCFA,然后跨膜进入乳腺上皮细胞参与乳脂合成,而参与LCFA跨膜运输的脂肪酸转运蛋白有：脂肪酸转运蛋白家族（Fattyacid transport proteins,FAPTs）、脂肪酸转位酶（Fatty acid translocase,FAT／CD36）、脂肪酸结合蛋白家族（Fattyacid binding proteins,FABPs）和小窝蛋白（Caveolinproteins）。文章在综述LCFA跨膜运输研究的基础上,重点介绍4种LCFA转运蛋白的研究进展,发现在奶牛乳腺中表达的只有FAT／CD36和FABPs,而FATPs和小窝蛋白在奶牛乳腺中是否表达并参与LCFA的转运仍有待进一步研究确定。因此,应利用营养学、分子生物学和细胞生物学等方法,在体内和体外对FAT／CD36、FABPs、FATPs和小窝蛋白在泌乳期奶牛乳腺中的表达规律、作用机理进行深入研究,以探明其在乳腺LCFA转运中的作用及调控机制．并为优化乳脂组成实施的营养调控提供科学依据。     

两歧双歧杆菌PRL2010细胞壁蛋白预测和功能分析

[目的]运用生物信息学方法预测两歧双歧杆菌PRL2010锚定于细胞壁的蛋白和功能分析,为后续研究该菌与宿主相互作用的分子机制奠定基础。[方法]用Phobius和Signal P 4.0软件对两歧双歧杆菌PRL2010全基因组编码蛋白中细胞壁蛋白进行预测,同时采用COG(Cluster of Orthologous Groups of proteins)功能数据库对预测的细胞壁蛋白进行功能注释和聚类分析。[结果]两歧双歧杆菌PRL2010基因组1 706个编码蛋白中,28个蛋白含有细胞壁的锚定结构域。细胞壁蛋白功能分析结果显示,28个细胞壁蛋白中,21个蛋白没有功能注释,7个蛋白有功能注释,其中1个蛋白(exo-alpha-sialidase)参与碳水化合物代谢与转运,2个蛋白(bacteriophage endolysin和cell wall-associated protein)参与细胞壁和细胞膜的生物合成,2个蛋白(hypothetical protein BBPR_0440和Subtilisin family peptidase)参与蛋白质翻译后修饰,1个蛋白(beta-n-acetylhexosaminidase Nag Z)参与细胞内运输、分泌和囊泡运输,1个蛋白(metallophosphoesterase)功能只是预测的。[结论]两歧双歧杆菌PRL2010的大多数细胞壁蛋白功能未知,还需要在以后的研究中进一步分析和注释。     

乳蛋白磷酸化分析方法研究进展

蛋白质的磷酸化是一种可逆性的蛋白质翻译后修饰,在调节蛋白结构和功能方面起重要作用。乳蛋白质的磷酸化与牛奶的稳定性有关。采用蛋白组学方法和技术,在实现乳蛋白质组成鉴定的同时,又能分析蛋白质的磷酸化修饰位点。文章综述了磷酸化蛋白质的检测和富集分离技术及乳蛋白磷酸化修饰的研究进展。     

泌乳反刍动物乳腺的氨基酸代谢

 乳腺是合成乳汁的主要场所。为了提高反刍动物饲料转化效率、泌乳量和乳蛋白产量，就有必要把乳腺氨基酸代谢作为研究的基础。本文从氨基酸供应、乳腺对氨基酸的摄取、乳腺氨基酸的代谢、能量供应对乳蛋白合成的调节等4方面，对泌乳反刍动物乳腺的氨基酸代谢进行综述，为进一步深入研究乳腺代谢，调控乳产量、乳蛋白含量提供依据。     

Nicotine blocks the suckling-induced rise in circulating prolactin in lactating rats

In lactating rats the rapid suckling-induced release of pituitary prolactin into circulating blood is inhibited by a subcutaneous injection of nicotine. This treatment does not block the lesser "stress-induced" rise in prolactin in response to either. Although nicotine may impair milk production by its effect on prolactin release, it does not appear to block milk ejection from the lactating mammary gland.     

阴外动脉灌注氨基酸对奶山羊乳腺摄取乳成分前体物的影响

 【目的】研究阴外动脉灌注不同平衡模式的氨基酸对奶山羊乳腺摄取乳成分前体物（milk components precursors,MCP）的影响规律。【方法】选用2—3周岁,体重、产奶量相近、安装有阴外动脉和腹部皮下静脉血插管的关中奶山羊3只,由阴外动脉灌注不同平衡模式的氨基酸,在灌注前后分别采集动静脉血浆,测定乳蛋白前体物（milk protein precursors,MPP）、乳脂前体物（milk fat precursors,MFP）和乳糖前体物（milk lactose precursors,MLP）的含量和乳腺对其的摄取。【结果】阴外动脉灌注不同平衡模式混合氨基酸改变了乳腺对MPP、MFP的摄取（P＜0.05）,乳腺对MLP的摄取理想模式组和平衡模式组均高于不灌注组,但理想模式组与不灌注组差异不显著（P＞0.05）,平衡模式组与不灌注组差异显著（P＜0.05）;不同氨基酸模式灌注组产奶量和乳成分明显高于对照组（P＜0.05）。【结论】阴外动脉中氨基酸的平衡改变了乳腺对乳成分前体物的摄取,有利于乳的合成。本试验条件下,阴外动脉中必需氨基酸达到理想平衡（AABI=0.972）时乳腺对MCP的摄取效果最佳。     

3＇ Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

To analyze miR-139 target sites in 3＇ UTR of GHR gene in dairy cow mammary gland, a GHR 3＇ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy...     

乳脂合成前体对奶牛乳腺上皮细胞亚细胞蛋白质组学影响

向奶牛乳腺上皮细胞中添加β-羟基丁酸钠,利用双向电泳技术进行细胞亚细胞蛋白质组学方面的研究。结果表明,β-羟基丁酸钠对奶牛乳腺上皮细胞亚细胞蛋白表达有显著影响。Imagemaster6.0软件分析出4个表达上调的蛋白点,鉴定分别是ENO1、α-glucosidase、plastin3和ARPC2。通过实时荧光PCR检测以上蛋白在mRNA水平的表达差异,结果均有不同程度的提高,其中以ENO1、α-glucosidase及Arpc2提高显著(P<0.05)。研究所筛选出的蛋白,将有助于进一步揭示奶牛乳腺上皮细胞增殖和泌乳机制。     

The Effect ofAdministration of Rutin on Plasma Levels of Estrogen,Prolactin, Growth Hormone and Gene Expression of Their Receptors in Mammary Glands in Ovariectomized Rats

The development of mammary glands, endocrine hormone concentrations and the gene expression of related receptors were measured in ovariectomized virgin rats after adminstration of an estrogen-like plant extract, rutin. Thirty-two ovariectomized virgin Wistar rats were randomly assigned to 4 treatments with 8 animals each: gastric infusion of 2 mL normal saline per unovariectomized rat per day (Sham), gastric infusion of 2 mL normal saline per ovariectomized rat per day (Ova), gastric infusion of 60 mg rutin kg-1 body weight (BW) per ovariectomized rat per day (Ova+Rut), or intramuscular injection of 60 μg estradiol kg-1 BW per ovariectomized rat weekly (Ova+Est). Samples of blood and mammary glands were harvested to determine the levels of estrogen (E2), prolactin (PRL) and growth hormone (GH), and the gene expression of estrogen receptors (ER), prolactin receptors (PRLR) and growth hormone receptors (GHR) with radioimmunoassy (RIA) and RT-PCR technology, respectively. The E2 concentration in plasma and gland tissues from the rats of Ovx+Rut or Ovx+Est was higher than that of Ovx (P<0.05), but the plasma E2 concentration from the rats of Ovx+Rut was lower than that of Sham (P<0.05). The order of the PRL concentration in plasma and gland tissues was Ovx     

中草药饲料添加剂对大鼠泌乳及乳腺发育的影响

在基础催乳方中分别添加含有异黄酮雌激素的两种中草药葛根和黄芪,测定其对大鼠泌乳、仔鼠增重和乳腺发育的影响。结果表明:葛根 基础方和黄芪 基础方组的仔鼠增重显著高于对照组和基础方组(P<0.05),而葛根和黄芪组仔鼠增重差异不显著(P>0.05),且对照组和基础方组差异也不显著(P>0.05)。剥离泌乳大鼠腹部一侧乳腺,测定大鼠乳腺组织中DNA和RNA的含量,结果表明葛根组和黄芪组的DNA、RNA和RNA/DNA明显高于对照组,与基础方组差异不显著(P<0.05);葛根组和黄芪组的RNA/DNA显著高于对照组,而与基础方组差异不显著。