蛋氨酸对奶牛乳腺上皮细胞亚细胞蛋白质组的影响研究

为寻找奶牛乳腺上皮细胞亚细胞结构中与泌乳相关的重要蛋白质、从蛋白质水平揭示乳蛋白合成的调控机制,应用双向凝胶电泳技术分离体外培养的蛋氨酸处理组与正常组奶牛乳腺上皮细胞蛋白质,利用Image Maser 2D软件对图谱进行对比分析,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)和数据库检索鉴定,并采用实时荧光定量PCR技术在mRNA水平上验证2-DE结果。质谱鉴定出8个表达上调的差异蛋白质,大多数差异蛋白质的功能涉及细胞骨架构成、能量代谢等过程。这些差异点的发现为研究奶牛泌乳机理提供了有益的线索。     

奶牛乳腺上皮细胞核蛋白质组双向电泳方法的建立

蛋白质样品的双向电泳分离效果受各种试验条件的影响较大,针对不同来源的蛋白样品进行试验条件的优化可获得具有较高分辨率的双向电泳图谱。试验拟建立优化的奶牛乳腺上皮细胞核蛋白质双向电泳分离体系,按标准条件对蛋白质进行双向电泳分离,并对关键因素进行优化,采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。通过对试验条件的筛选和优化,成功建立了具有较高分辨率的奶牛乳腺上皮细胞核蛋白质组双向电泳分离体系。     

健康奶牛与临床型乳腺炎奶牛乳腺核蛋白组差异表达分析

亚细胞蛋白质组的优点在于对低丰度蛋白的研究,运用亚细胞蛋白质组学的研究策略,可以提高低丰度蛋白质在双向电泳中的检出数量.通过分析奶牛乳腺炎乳腺与正常乳腺核蛋白组差异表达情况,为奶牛乳腺炎发病机理研究寻找尽可能多的生物标记分子.经超速离心法分离细胞核,双向凝胶电泳分离蛋白,用PDQuest7.4软件分析寻找差异蛋白质斑点,高效液相色谱串联离子阱质谱鉴定蛋白质.从核蛋白组2-DE图谱中筛选出22个差异表达的蛋白质斑点,质谱鉴定出17个差异表达的蛋白质,2个蛋白质在乳腺炎发病过程中下调,7个上调,5个只在正常情况下表达,3个只表达在乳腺炎组织中,筛选出的差异表达蛋白质涉及细胞骨架构成、代谢调节及凋亡调控等许多方面.表明奶牛乳腺炎发生时乳腺组织核结构和代谢状态都发生了的变化.     

双向电泳分析蛋氨酸对奶牛乳腺上皮细胞核磷酸化蛋白质的影响

为了寻找奶牛乳腺上皮细胞核中与乳蛋白合成相关的重要蛋白质,从翻译水平揭示乳蛋白合成的机理,本试验应用双向凝胶电泳技术和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)的方法,分析添加蛋氨酸后奶牛乳腺上皮细胞核磷酸化蛋白质的变化,并采用Western blotting验证。质谱分析鉴定出5个表达上调的细胞核磷酸化蛋白质,分别是葡萄球菌核酸域包含蛋白质1、甘氨酰-tRNA合成酶、Septin-6、Twinfilin-1和延长因子1-beta,这些蛋白质的功能涉及DNA转录、mRNA翻译、蛋白质合成、细胞分裂、细胞形态发生及细胞周期的调控。     

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。     

双向电泳检测奶牛乳腺上皮细胞核磷酸化蛋白质方法的建立

为研究不同激素或营养素对乳腺上皮细胞核磷酸化蛋白质的影响,本实验建立细胞核磷酸化蛋白质提取方法及双向电泳实验技术。结果表明,细胞核磷酸化蛋白纯化结合双向电泳技术是乳腺上皮细胞核磷酸化蛋白质组学研究的有效方法,可为进一步研究乳腺上皮细胞机理提供技术基础。     

基于二维凝胶电泳分析健康泌乳奶牛血浆蛋白质组的个体差异

为了分析奶牛血浆蛋白质存在的个体差异,以合理选择牛血浆蛋白质组研究中试验奶牛的样本数。试验采集了临床健康的泌乳早期的初产中国荷斯坦奶牛的血液,分离血浆蛋白后用二维凝胶电泳技术分离,考马斯亮蓝G-250染色后,对分离形态好的56个蛋白点的表达丰度进行分析。结果发现,蛋白点的变异系数在8.7%～59.6%之间,且有5个蛋白点的丰度变异系数较大（35%～59.6%）,用液相色谱串联离子阱质谱分析后,3个蛋白点获得有效鉴定,为转甲状腺素蛋白、载脂蛋白A1和α1酸性糖蛋白。方差分析结果表明,当样本数≥4时可有效降低组内样本间的个体差异。因此,奶牛血浆蛋白的表达存在个体差异,本试验为奶牛血浆蛋白质组研究中合理选择样本数量提供了依据。     

乳腺炎奶牛乳腺肥大细胞的改变

乳腺炎是奶牛最重要的感染性疾病之一，它不仅直接危害奶牛健康，影响奶的质量、降低奶牛繁殖性能，而且由于应用大量抗生素治疗乳腺炎而出现的“含抗牛奶”，已成为严重危害人们食用安全的污染食品。Sordilo等曾用白介素-2(IL-2)对奶牛乳腺炎进行治疗研究，但由于迄今为止尚不清楚IL-2与乳腺炎发病机制的关系，其疗效也不理想。而肥大细胞作为     

奶牛乳腺功能研究的生物材料来源分类及其特征比较

奶牛乳腺是一个具有复杂生物结构、多种类细胞群系和具有分泌功能的器官,因此乳腺功能研究材料可以来源于实体组织获取和乳腺分泌物质等多种方式。基于不同试验目的,需要根据现有理论依据来选择相应的乳腺生物材料。本文论述了奶牛乳腺结构及其与所获得生物材料分类的多样性,总结了利用不同乳腺生物材料在多层次生物学探究（转录组、蛋白组、代谢组）中的应用价值及特征,为奶牛乳腺功能、原料乳品质优化和幼龄反刍动物健康调控方面的研究提供重要参考。     

雌激素处理的奶牛乳腺上皮细胞核磷酸化蛋白质组学分析

雌激素调节多种生理过程,包括乳腺生长发育、形态发生、增殖和分化及乳蛋白的表达。许多磷酸化蛋白调节乳蛋白合成,如磷酸化Stat5和mTOR,但在乳蛋白合成的转录和翻译后水平的调节鲜有报道。为了阐述雌激素在奶牛乳腺上皮细胞中的作用机制,本试验利用双向电泳和质谱分析,鉴定了雌激素调节的细胞核磷酸化蛋白质。在雌激素处理24h后,鉴定出7个表达上调的蛋白质,包括以前报道过的甘氨酰-tRNA合成酶,属于氨基酰-tRNA合成酶家族Ⅱ;这些蛋白质涉及翻译起始因子、GTP结合的核蛋白、热休克蛋白、泛素-蛋白酶体系统。这些筛选出来的蛋白质,揭露了雌激素影响奶牛乳腺上皮细胞核磷酸化蛋白质,为进一步研究乳合成的分子机制提供一个新的途径。     

Effects of stocking density on oxidative stress status and mammary gland permeability in early lactating dairy cows

The current study was conducted to investigate the effect of stocking density (SD) on oxidative stress status and mammary gland permeability in early lactating dairy cows. Thirty‐two dairy cows were allocated into 16 blocks, basing on parity, previous milk yield, and body weight, and were then randomly assigned into one of the two treatments as follows: 75% (75SD) and 100% (100SD) SD. The cows were fed with same diet throughout the 8‐week experimental period. The milk yield and milk sample were collected on two consecutive days during the 8‐week experimental period weekly. Plasma samples were collected on fourth and eighth experimental weeks. Raw, energy‐corrected, and 4% fat‐corrected milk yield were significantly higher in 75SD‐cows than that of 100SD‐animals, respectively. The milk somatic cell count was lower in 75SD‐cows than that of 100SD‐animals. The levels of Na⁺, Na⁺/K⁺, bovine serum albumin and plasmin were lower in 75SD‐cows than those of 100SD‐cattle, respectively. The 75SD‐cows had reduced insulin and insulin/glucagon levels but higher prolactin and growth hormone concentrations, compared with those of 100SD‐animals, respectively. In conclusion, compared with low SD‐animals, early lactating cows with higher SD had higher oxidative stress status, which further led to a greater mammary gland permeability.     

蹄叶炎奶牛血浆蛋白质组学2-DE图谱的构建及分析

采集患病牛和健康牛血浆,用人白蛋白和IgG抗体去除其高丰度蛋白后,通过双向凝脉电泳(Two dimension-al gel electrophoresis,2-DE)技术和PDQuest分析软件比较2组间血浆蛋白组表达差异.结果显示,蹄叶炎奶牛血浆2-DE图谱中有39个蛋白斑点表达上调,23个蛋白斑点表达下调,共检测到62个差异蛋白点.结果表明,奶牛发生蹄叶炎时,其血浆蛋白组表达模式发生了改变,这种改变可能与蹄叶炎的发病机制密切相关.     

健康与患布鲁菌病奶牛血清蛋白2-DE的建立和初步分析

采用商业化试剂盒Albumin & IgG Depletion Spintrap处理奶牛血清样品,比较处理前后对2-DE图谱的影响,结果发现处理后奶牛血清2-DE图谱中高丰度蛋白明显减少,但低丰度蛋白也有损失.采用不去除高丰度蛋白的方法比较健康奶牛与感染布鲁杆菌病奶牛的血清2-DE图谱,发现了11个明显差异的蛋白点,其中3个在正常奶牛血清中高表达,1个只在正常奶牛血清中表达;6个在患布鲁菌病奶牛血清中高表达,1个只在患布鲁菌病奶牛血清中表达.这些差异蛋白点的发现为将来质谱分析奠定基础.     

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

青贮玉米秸饲喂奶牛效果研究

采用在奶牛日粮中分别加入青贮玉米秸和玉米秸对比试验的方法，经过30d锔喂试验，表明试验组比对照组日均产奶高1．24kg,差异显著（P＜0．05）。     

Secretory carcinoma of the mammary gland in an 8-year-old Holstein-Friesian dairy cow

This study was conducted to examine the oxidative stress biomarkers in a cow diagnosed with a follicular cyst in her left ovary. Progesterone (P4) and plasma oxidative stress status was measured in 13 Holstein cows after synchronization of oestrus with controlled internal drug release (CIDR) and prostaglandinF2α (PGF2α) protocol. The presence and size of ovarian structures were monitored by transrectal ultrasound at 4 hourly intervals. Of the 13 cows, 12 were monitored until ovulation was detected and recorded, whereas one cow failed to ovulate and developed a follicular cyst. Oxidative stress biomarkers; reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP), oxidative stress index (OSI), glutathione (GSH), ceruloplasmin and advanced oxidation protein products (AOPP) were measured in the cystic cow and compared to those of the 12 ovulated cows and are referred to as higher or lower if they are outside the mean ± standard error of mean of those of ovulated cows. The cystic cow had lower ROMs and OSI between 36 and 84 h after PGF2α injection and at 9 h, from 36 to 60 h after PGF2α injection respectively. On the other hand, antioxidant (BAP and GSH) was higher in the cystic cow compared to her ovulated herd mates. The observed imbalance between oxidant and antioxidant might have disrupted the physiological events for ovulation to occur, leading to cystic ovarian disease.     

灌注葡萄糖对奶山羊乳腺营养摄取的影响

本试验研究了阴外动脉灌注葡萄糖对乳腺营养物质吸收和乳成分的影响。选用体重和产奶量相近的4只泌乳中期关中奶山羊,做阴外动脉和腹部皮下静脉手术安装血插管,采用4×4拉丁方设计,每天分别灌注葡萄糖0、12、24g和36g。结果表明:乳脂率随葡萄糖灌注量的增加而显著降低(P0.01)。灌注36g/d处理组乳脂产量显著低于其他处理组(P0.01);葡萄糖灌注提高了奶山羊的产奶量,24g/d处理组的产奶量显著高于对照组(P0.01);36g/d处理组乳腺对葡萄糖的摄取量和乳糖的产量都显著高于对照组(P0.01)。甘油三酯和非脂固形物的含量各组间差异不显著(P0.05),但葡萄糖灌注提高了乳蛋白产量,其中24g/d处理组乳蛋白产量最高,并显著高于对照组(P0.05)。葡萄糖的灌注显著降低了奶山羊干物质的采食量(P0.01)。葡萄糖的灌注对乳腺氨基酸的摄取影响不显著。葡萄糖的灌注使氨基酸合成乳蛋白的产出率提高1%～9%,葡萄糖合成乳糖的产出率提高0%～5%,有效改善了乳成分。