Close association of gonadotropin-releasing hormone fibers and gonadotropin, growth hormone, somatolactin and prolactin expressing cells in pejerrey, Odontesthes bonariensis

The purpose of this study was to determine if there is any association between immunoreactive (ir) gonadotropin-releasing hormone (GnRH) fibers with different pituitary endocrine cell types in the pejerrey, Odontesthes bonariensis. Using a monoclonal antibody raised against mammalian GnRH (mGnRH) (LRH13), ir-GnRH fibers were observed passing through the pituitary stalk and reaching the three areas of the pituitary gland: rostral (RPD) and proximal pars distalis (PPD) and pars intermedia (PI). Double labeled immunocytochemistry showed ir-GnRH fibers in close association with prolactin (PRL)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropin (GtH)-producing cells in the PPD and the external border of the PI, and with somatolactin (SL)-producing cells in the PI. Our results show, direct morphological evidences of a close association of GnRH fibers with GH, PRL, GtH and SL-expressing cells. These results would suggest that GnRH has a broad role in the regulation of the secretion of different pituitary hormones.     

Characterization of dual enzyme resulted from bicistronic expression of two β-glucanases in porcine cells

Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four arti?cial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only pBgA and pEgx showed high activity in transfected pig kidney cells. To improve the pH range and pH stability of β-glucanase, the two β-glucanases, pBgA and pEgx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of pBgA3pEg and pBg2ApEg showed significantly enlarged pH range and significantly increased pH stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.     

Effects of dietary protein sources on growth,survival and digestive capacity of Octopus maya juveniles (Mollusca: Cephalopoda)

We propose two hypotheses to explain the inexistence of adequate prepared diet for Octopus maya at this date: Hypothesis 1 is related to changes in protein structure during protein cooking, which affects the digestibility. Hypothesis 2 is related to changes on nutritional characteristics during ingredient process, which affects the nutritional composition of diet. To test hypothesis 1, experiments one and two were directed to determine if protein cooking reduces digestibility and growth of animals when compared to fresh or lyophilized protein sources. For hypothesis 2, three experiments were conducted, testing seven different dietary protein sources offered in isolation or combined in artificial diets fed to O. maya. Results demonstrated that the diets that promoted growth were the ones based on fresh crab paste, and both lyophilized crab and squid tentacles paste. In consequence hypothesis 1 was accepted. The cooking process also changed nutritional characteristics of protein sources, affecting the growth of O. maya. Results obtained when squid and crab were mixed suggest that nutritional requirements of octopuses were covered with that diet in similar forms compared to when using fresh or lyophilized crab, also confirming hypothesis 2. Based on growth rates obtained, we can conclude that nutritional requirements of O. maya must be between 80% and 86% Protein (P), 5.1–5.6% Lipids and a protein: energy ratio between (P/E) 38.9 and 42.2 g MJ^?1.     

不同浓度胰岛素样生长因子-Ⅰ对体外培养奶牛乳腺上皮细胞乳蛋白和脂肪合成相关基因表达的影响

应用Real-time PCR方法检测不同浓度(0、1、10和100 ng/mL)胰岛素样生长因子-Ⅰ(IGF-Ⅰ)处理后的体外培养奶牛乳腺上皮细胞乳蛋白和脂肪合成相关基因mRNA的相对表达量。结果表明,添加不同浓度IGF-Ⅰ对体外培养的奶牛乳腺上皮细胞IGF-Ⅰ受体基因(IGFIR)、IGF-Ⅰ结合蛋白-3基因(IGFBP3)、α-s1-酪蛋白基因(CSN1S1)和κ-酪蛋白基因(CSN3)mRNA的相对表达丰度均无显著影响(P>0.05)。随着IGF-Ⅰ添加浓度的增加,β-酪蛋白基因(CSN2)、乙酰辅酶A羧化酶基因(ACACA)、脂肪酸合成酶基因(FASN)和脂肪酸结合蛋白-3基因(FABP3)mRNA的相对表达丰度显著上调(P<0.05)。提示,IGF-Ⅰ作为一种重要细胞因子参与调节乳腺上皮细胞乳蛋白和乳脂肪相关基因mRNA的表达。     

人凝血因子Ⅸ乳腺特异表达载体的构建及细胞转染

【目的】人凝血因子Ⅸ（human coagulation factor Ⅸ,hFIX）是凝血过程中的关键因子,对临床治疗血友病有着极其重要的意义。研究旨在构建人凝血因子Ⅸ乳腺表达载体,在猪乳腺上皮细胞中验证其乳腺特异表达能力,获得hFIX转基因雌性乳腺上皮细胞和猪胎儿成纤维细胞,为克隆乳腺特异表达人凝血因子Ⅸ的转基因猪做准备。【方法】按照TaKaRa公司RNAiso Reagent和Prime Script RT-PCR试剂盒的说明书,从人胎儿肝脏中提取RNA并扩增出人凝血因子Ⅸ（human coagulation factor Ⅸ,hFIX）cDNA序列,利用PCR方法扩增牛生长激素(BGH) polyA序列,将hFIX的cDNA序列与BGH polyA序列分别连接到表达载体pbCSN2-RC的牛酪蛋白（CSN2）启动子之后,构建带有新霉素抗性和红色荧光蛋白双筛选标记的乳腺特异性表达载体pbCSN2-hFIX-pA-RC。取本地屠宰的泌乳期猪乳房组织,利用组织块种植法培养猪乳腺细胞,并通过控时消化分离纯化猪乳腺上皮细胞,进行染色体分析后,挑选含有正常染色体数目的细胞,通过脂质体法将表达载体导入,经过G418筛选获得稳定表达红色荧光蛋白的转基因细胞,反转录、实时定量PCR验证乳腺表达载体构建的合理性和有效性。为了只对雌性胎儿成纤维细胞进行转基因操作,首先对培养的猪胎儿成纤维细胞进行SRY基因PCR扩增,确定其性别。之后,利用脂质体将证明有效的表达载体导入细胞,并进行G418和红色荧光蛋白表达筛选,对经过鉴定的阳性细胞进行扩大培养和冷冻保存,为下一步的体细胞克隆做准备。【结果】经PCR和酶切鉴定,人凝血因子ⅨcDNA和BGH PolyA成功克隆到载体pbCSN2-RC中,获得具有双筛选标记的pbCSN2-hFIX-pA-RC。转染后G418和红色荧光蛋白表达阳性的猪乳腺上皮细胞,经实时定量PCR鉴定,hFIX有高效表达,证明所构建载体具有指导人FIX在乳腺特异表达的能力,可以用于生产乳腺特异表达的动物。将构建的载体转入雌性猪胎儿成纤维细胞,经G418和红色荧光蛋白的表达筛选,成功获得hFIX转基因阳性细胞。【结论】pbCSN2-RC所含的牛β-酪蛋白启动子可以成功特异性诱导hFIX在猪乳腺细胞的特异表达,获得的hFIX转基因阳性猪胎儿成纤维细胞,为下一步通过体细胞克隆培育hFIX乳腺生物反应器奠定了基础。     

柴芍乳癖颗粒对乳腺增生兔模型B超声像及组织形态学的影响

目的 观察柴芍乳癖颗粒对乳腺增生病兔模型乳腺组织B超声像及组织形态学的影响。方法 56只健康未孕新西兰雌兔,随机挑出10只为正常对照组,肌注生理盐水;46只为模型制备组,序贯肌注雌孕激素造模。2组均随机处死1只雌兔,观察病理切片确定造模成功后,将模型制备组剩下的45只兔再随机分为5组：模型对照组、乳癖散结颗粒组、柴芍乳癖颗粒低、中、高剂量组。6组兔每天分别予以生理盐水、乳癖散结颗粒（0.56 g/kg）、柴芍乳癖颗粒（0.47、0.94、1.88 g/kg）水溶液灌胃1次,连续3月。末次灌胃 24 h后麻醉,用B超机采集第2对乳房的声像图,处死后取第2对乳腺组织,HE染色观察组织形态学表现,计数乳腺小叶腺泡数、导管上皮细胞层数、腺泡腔直径。结果 柴芍乳癖颗粒能使模型兔乳腺组织低回声区缩小,内部回声强度减低,其中高剂量组最明显;与模型组相比,柴芍乳癖颗粒低、中、高剂量跟乳癖散结颗粒均可明显缩小腺泡腔直径（P<0.01）,中、高剂量可以减少乳腺导管上皮细胞层数,低剂量还可减少乳腺小叶腺泡数（P<0.05）。结论 柴芍乳癖颗粒可以改善乳腺增生兔模型B超声像,减轻雌兔乳腺增生程度,治疗乳腺增生效果明显。     

奶牛SREBP1蛋白在乳腺上皮细胞的表达定位及对SCD1基因启动子的转录调控

【目的】固醇调节元件结合蛋白1(SREBP1)作为核转录因子,对于细胞脂肪合成酶基因的表达发挥着重要的调控作用。论文旨在奶牛乳腺上皮细胞中研究SREBP1对于SCD1基因启动子的转录调控作用,为进一步明确SREBP1对于靶基因的转录调控机制提供理论基础。【方法】以荷斯坦奶牛乳腺组织的c DNA为模板,采用分段克隆的方法获得SREBP1基因的编码序列,通过重组酶与pc DNA3.1载体进行重组环化构建pc DNA3.1-SREBP1表达载体,将构建的载体测序验证后提取质粒,转染奶牛乳腺上皮细胞。以EIF3K基因为内参基因,采用荧光定量PCR检测SREBP1基因m RNA的表达差异;采用免疫荧光的方法对SREBP1进行标记,以DAPI复染细胞核,激光共聚焦观察SREBP1蛋白的亚细胞定位;转染含有不同调控元件的SCD基因启动子,同时转染1.0μg pc DNA3.1-SREBP1作为处理,荧光素酶报告基因系统分析启动子活性;分别转染0.25、0.5和1μg的pc DNA3.1-SREBP1载体,分析p GL3-SCD 2和p GL3-SCD3启动子活性与SREBP1之间的量效关系。【结果】分段克隆得到的PCR产物分别为1 170、1 116、363和900 bp的片段,经过与pc DNA3.1载体重组后获得pc DNA3.1-SREBP1表达载体,经酶切和测序验证,发现除1个无义突变外,与标准序列完全相同,整个序列长度达到3 510bp;将pc DNA3.1-SREBP1载体转染乳腺上皮细胞后,Real-time PCR检测发现与转染空载体的对照组相比,SREBP1基因的m RNA表达倍数增强130.4倍(P0.001);激光共聚焦观察发现,DAPI染色的细胞核呈蓝色,免疫荧光标记的SREBP1呈绿色,二者融合后呈现青色,共定位在乳腺上皮细胞核中;启动子活性检测发现,与p GL3-SCD1、p GL3SCD 2相比,SREBP1处理能够极显著增加p GL3-SCD3、p GL3-CD4启动子的活性(P0.001),分别比对照组提高了1.0倍和0.7倍,进一步分析发现,在用0.25—1μg的pc DNA3.1-SREBP1处理后,与p GL3-SCD2的启动子活性持续下降相比,p GL3-SCD3的启动子活性从59.81上升到108.43(P0.001),二者存在剂量效应关系,结合SCD2和SCD 3启动子上主要的结构差异SRE元件(5′-AGCAGATTGCG-3′),推测此序列可能是SREBP1调控SCD基因启动子转录的结合序列。【结论】克隆构建奶牛SREBP1基因表达载体,亚细胞定位SREBP1蛋白主要在乳腺上皮细胞核中,SREBP1可以与SRE调控元件结合促进SCD1基因启动子的转录。     

黄野螟性信息素分泌腺形态及超微结构

为深入了解黄野螟Heortia vitessoides性信息素生物合成及释放机制,采用光学显微镜、扫描电镜和透射电镜对黄野螟雌成虫性信息素分泌腺形态和超微结构进行观察。结果表明,黄野螟雌成虫性信息素分泌腺为一个完整的环状上皮结构,位于腹部末端第8腹节和第9腹节之间的节间膜上。黄野螟雌成虫性信息素分泌腺表面分布着3类8种感器,即I～IV型毛形感器、I和II型刺形感器及I和II型锥形感器。分泌腺细胞单层密集排列在角质层内表面,细胞呈柱状或圆形,表皮层分为上表皮和内表皮,细胞中还有发达的肌肉层、脂滴以及线粒体等结构组织。     

一例犬乳腺导管乳头状癌的诊断与治疗

犬乳腺导管乳头状癌是犬乳腺肿瘤的一种,为恶性肿瘤,多发于中老年未绝育母犬。临床症状为乳腺出现肿块,大小不等,生长快速,肿瘤可以在一个或多个腺体中发展。在晚期转移的情况下,癌变会扩散到淋巴结,肺,结肠,肾脏,有时还会扩散到骨骼,及早施行手术通常预后良好。本文对一例8岁母犬的乳腺导管内乳头状癌病例的临床诊断与手术治疗情况作详细介绍,以期为犬乳腺肿瘤的临床诊治提供参考。     

奶牛乳腺的防御机制与乳腺炎病理学

乳头管的结构和内衬的角质蛋白是乳腺抵御微生物侵入的物理性屏障,角蛋白中的低级脂肪酸、阳离子蛋白质对侵入的病原微生物具有抗性;环境性致病菌是引起机体免疫应答的主要因子,巨噬细胞、单核细胞、中性粒细胞和淋巴细胞之间的相互协调组成了自然的免疫系统,它们通过分泌细胞因子、抗体和吞噬作用等活动来灭杀、清除侵入机体的病原体;乳中的可溶性因子具有免疫调节作用,对细菌的结构具有破坏性;妊娠期及围产期奶牛由于内分泌的变化而导致免疫抑制,结果造成临床型和亚临床型乳腺炎的发病率相对较高;牛奶体细胞计数对亚临床型乳腺炎的监控具有参考性。