Persistence of gentamicin residues in milk after the intramammary treatment of lactating cows for mastitis

This study was designed to investigate persistence of gentamicin residues in milk after the intramammary treatment of lactating cows for mastitis. Milk samples were collected at a 1-d interval after the last administration from 34 individual cows that had received intramammary infusions of gentamicin. The doses and treatment times evaluated in this study represented those that have been applied by veterinarians in practice. The tetrazolium chloride assay was used to determine whether there were significant residues of the antibiotic in the samples. Persistence of detectable drug residues in milk from 33 cows (28 cows, ≤6 infusions at ≤0.7 g gentamicin; and 5 cows, 2 infusions at 0.8 g gentamicin) did not exceed 5 d; but 1 cow (5 infusions at 0.8 g gentamicin) had detectable residues in its milk for 9 d. Our results suggest that a 5-d milk withdrawal period might be insufficient to secure the clearance of the contamination of gentamicin, because treatment times and dosages contribute to the antibiotic clearance. A larger scale of samples are needed for further investigations.     

铁母散合剂对杂交奶牛乳房炎的预防和治疗效果观察

采用铁苋菜、地锦草等中草药组成铁母散合剂.对杂交奶牛临床型乳房炎和隐性型乳房炎进行预防和治疗试验.结果表明,铁母散合剂对西杂奶牛临床型乳房炎的治愈率均为80%,有效率均为100%;对隐性型乳房炎的治愈率和有效率均为100%,对干奶期乳房炎的预防效果,其发病率均只有20%,而生理盐水对照组的发病率却高达60%.采用铁母散合剂对西杂奶牛乳房炎进行治疗后,两组乳汁中均未检出有病原菌.两组乳中体细胞数均降至正常水平(50万个/mL以下).乳清中的白蛋白(SA)、乳酸脱氢酶(LDH)、天门冬氨酸氨基转移酶(AST)及丙氨酸氨基转移酶(ALT)等各项生化指标均显著降低至正常水平.而采用青、链霉素治疗的两组乳汁中各有一头检出有大肠杆菌.两组乳中体细胞数均未降至正常水平.乳清中的(SA)、(LDH)、(AST)(ALT)等各项生化指标亦未能降低至正常水平.本次试验结果表明:铁母散合剂对西杂奶牛乳房炎的防治效果优于抗生素.     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.     

奶牛金黄色葡萄球菌乳腺炎小鼠模型的建立

目的利用从临床乳腺炎奶牛乳样中分离纯化的金黄色葡萄球菌（S.aureus）感染小鼠乳腺,建立奶牛金黄色葡萄球菌乳腺炎小鼠模型。方法将产后8～10 d的ICR远交系母鼠随机分为3组,包括无菌生理盐水对照组,低剂量攻菌组（含金黄色葡萄球菌2×102CFU）和高剂量攻菌组（含金黄色葡萄球菌5×106CFU）。以小鼠第4对乳腺为攻菌部位,用非损伤方法经乳头管注入生理盐水或金黄色葡萄球菌。攻菌24h后采集乳汁进行白细胞计数;从眼球采血,进行血常规检测和流式细胞计数;之后处死母鼠,取乳腺组织进行匀浆细菌计数,并做乳腺组织切片。结果高剂量攻菌组不论是体温、组织学观察,还是乳汁白细胞计数、乳腺匀浆细菌计数、血常规指标和流式细胞计数,都和对照组有显著或极显著差异（P〈0.05）;而低剂量攻菌组与对照组相比,除血液CD4＋/CD8＋比值差异显著外（P〈0.05）,其余指标差异均不显著。结论金黄色葡萄球菌浓度为5×106CFU、攻菌时间24 h时,成功建立了远交系小鼠金黄色葡萄球菌乳腺炎模型,而2×102CFU细菌浓度则不适于远交系小鼠乳腺炎模型的建立。     

丸红活菌制剂(BLCS)饲喂奶牛试验研究

奶牛生产中饲喂丸红活菌制剂(BLCS),可明显提高奶牛的生产性能,且奶牛体况健康,毛管油亮,可预防乳房炎、子宫炎等生殖疾病发生,而且生产性能显著提高.选择52头新西兰荷斯坦奶牛随机分为两组,每组26头,试验组添加丸红活菌制剂(BLCS),结果表明:试验组比对照组提高产奶量1.29kg/头·天(p＜0.01),干物质提高了0.85%,乳脂率提高0.18%,乳蛋白提高0.09%.     

乳牛隐性乳腺炎诊断液——JMT临床应用效果及与其它方法的对比试验

采用新型研制的吉林乳牛隐性乳腺炎诊断液(JMT)对1173头乳牛的4560乳区进行了隐性乳腺炎的诊断,并与LMT-r、N.F.T法作比较,其结果表明JMT-s的检出率最高。同时从受检乳样中,随机选择214份(乳区)进行乳汁体细胞计数,以其作为基准,检验JMT-s、JMT-z、LMT-r及N.F.T等四种方法的准确性,结果JMT-s的符合率亦为最高(83.78％)。JMT试液采用国产原料配制,来源可以得到保证,价格低廉,适于推广。     

新疆北疆地区临床型乳房炎病原细菌的分离鉴定

旨在调查新疆北疆地区奶牛乳房炎高发季节细菌性乳房炎病原菌种类,为该病的药物防治及免疫学防治提供科学依据。选择新疆北疆14个规模化牛场共采集623份临床型乳房炎奶样进行了细菌分离鉴定,结果显示,分离得到468株细菌,其中兼性厌氧革兰氏阴性杆菌占25．2％;链球菌占22．6％;葡萄球菌和微球菌52．2％。     

牛注射猪瘟弱毒苗后对其安全性和产奶量的研究评估

用不同剂量的猪瘟乳兔组织苗给牦牛、奶牛、怀孕奶牛、小犊牛皮下注射,观测15天,并与注苗前和对照组比较,结果表明,受试牛注苗后,精神、饮食、体温、血相正常,妊娠母牛无一出现流产、难产、和死胎,注苗后产奶量与注苗前产奶量无显著差异.可见,猪瘟弱毒苗对牛是安全可靠的,注苗后不影响产奶量,该项研究为将来用猪瘟防制牛黏膜病奠定了基础.     

用电泳和高效液相色谱法检测水牛乳中掺入的普通牛乳

本研究的目的是建立检测水牛乳中掺入普通牛乳的方法．在德昌水牛乳中添加不同比例的普通牛乳,用电泳或高效液相色谱分析乳清蛋白组成,根据乳中β-乳球蛋白在水牛与普通牛之间的差异,可准确鉴定水牛乳中掺入的普通牛乳．聚丙烯酰胺凝胶电泳方法能检测到掺入的1％的普通牛乳;反相高效液相色谱法能在30min内检测到掺入的10％的普通牛奶．本研究为水牛乳的质量监控提供了可行的技术手段．     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

High expression of human serum albumin in milk of trans- genie mice directed by the goat p-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene pro-moter, 5'upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5 , 3 ) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

大鼠肝库普弗细胞的分离、鉴定及纯度、活性分析

按Higgins等方法制作大鼠2／3肝切除（partial hepatectomy,PH）模型,用两步灌流法分散肝脏细胞,用60％Percoll梯度离心结合免疫磁珠方法分离库普弗细胞（Kupffer cell,KC）,用外胚层发育不良抗原2（ectodermal dysplasia antigen2,ED2）和溶菌酶（lysozyme,LYZ）的免疫组织化学方法定性、定位再生肝（regenerating liver,RL）、分散的肝脏细胞及分离的库普弗细胞,用RT—PCR定量库普弗细胞的ED2和LYZmRNA,用蛋白免疫印迹方法定量库普弗细胞的ED2和LYZ蛋白．初步证实,分离的肝库普弗细胞中ED2和LYZ阳性细胞占96％以上,从PH后各时间点分离的库普弗细胞ED2和LYZmRNA量稳定,相应的蛋白量亦稳定。表明改进的分离库普弗细胞方法具有收率和纯度高、活性好等优点,值得采用．     

奶样无乳链球菌PCR检测方法敏感性研究

意义无乳链球菌是引起牛临床或亚临床乳腺炎主要病原菌,研究PCR奶样检测方法的敏感性,对探索病原无乳链球菌的简易、快速诊断具有重要意义.目的通过对含细菌数不同奶样的PCR扩增试验的观察和比较研究,探索其检测方法的敏感度.方法常规检测方法初选奶样,然后对其病原菌奶样进行培养、增菌和生化鉴定,筛选出牛无乳链球菌.将得到的无乳链球菌按10个稀释度奶样进行稀释,以标准该菌株作为阳性对照,分别进行PCR扩增、电泳.结果基于编码16S rRNA亚基的编码基因序列设计引物的PCR方法具有独特敏感性,可以检测到1ml生奶样中的一个细菌.     

针刺得气研究现状及思考

 针灸疗效得到世界公认，但如何传承数千年针灸的治疗经验和标准化针灸刺激手法，尤其是临床上如何确定有效的针灸刺激量，成为针灸研究、应用，乃至向世界传播的难题。基于中医传统理论和针刺得气的现代研究进展，思考了得气理论的意义，并提出得气是针灸取得最佳疗效的一个重要前提，在研究如何充分发挥针灸治疗作用及评价针灸疗效时，得气是不可或缺的重要参数的见解。针灸临床试验应进一步纳入得气的客观量化评价体系，科学地探索得气现象背后的生物学机制，阐明得气与疗效之间的关系。     

针刺治疗女性生殖障碍研究现状

 针刺作为一种传统疗法,在中国治疗妇科疾病有着悠久历史。20世纪80年代,针刺治疗女性生殖系统疾病在西方医学也确立了合法地位,其学术成果现在已经为国际学术界公认。本文概述了针刺在女性生殖障碍领域应用的国内外研究现状,包括国际最新研究进展和发展趋势、国内研究现状和水平,尤其是针刺在体外受精-胚胎移植和多囊卵巢综合征中的应用,包括针刺对体外受精-胚胎移植（IVF-ET）中胚胎着床及妊娠率的影响,针刺治疗PCOS机制研究,针刺对PCOS促排卵的临床疗效;分析国际竞争态势,分析国内研究落后的现状及存在问题;展望国内今后研究的发展方向。     

针刺留针时间对缺血性中风患者脑血流速度的影响

目的:观察不同留针时间对缺血性中风患者脑血管血流速度的影响,以探索针刺治疗缺血性中风的时效关系的机理。方法:选取缺血性中风急性期、恢复期的患者34例,按轻、中和重度随机分成留针20、40和60 m in 3组。在针刺治疗的疗程前后,对患者进行经颅多普勒检测。结果:针刺可提高缺血性中风患者脑血管血流速度,以留针60 m in作用最为明显。结论:针刺留针时间与脑血管血流速度之间有一定的时效关系。     

牛酪蛋白基因导入大豆的研究

将带有牛酪蛋白基因caseinB的植物表达载体 pAS -2 ,在大豆自花授粉后 ,用注射法导入大豆受精子房中 .以质粒 pAS -2的 3 5S -Casein -Gus片段为模板 ,随机引物法标记探针 ,对 1 0 3株受体植株后代的DNA进行点杂交和Southern杂交 ,发现其中 1株在点杂交和Southern杂交中均呈阳性 .证明外源基因已整合到大豆基因组中 .     

人白介素-3乳腺表达载体的构建

人白介素-3是一种造血系统和免疫系统调节剂,在治疗造血系统疾病、肿瘤、先天或获得性免疫功能缺陷等方面发挥着重要作用.应用牛乳腺生物反应器生产人白介素-3的研究具有重要的临床应用和经济价值.研究选用具有红色荧光蛋白报告基因(R ed2)和新霉素抗性基因(neor)表达框架的pD sR ed2-1质粒为骨架构建人白介素-3乳腺表达载体.通过PCR方法分别扩增牛β-酪蛋白基因5′端上游调控序列、人IL-3基因以及CM V启动子序列,将它们按先后顺序分别定向克隆于质粒pD sR ed2-1的多克隆位点内,使牛β-酪蛋白基因调控序列位于人IL-3基因的上游,指导人IL-3基因在乳腺组织中特异性表达,而CM V启动子位于红荧光蛋白基因的上游,指导红荧光蛋白基因在所有的组织中非特异性表达.限制性酶切片段分析及部分DNA序列鉴定结果表明,所构建载体结构正确.     

HPLC-MS/MS测定大鼠血浆中杨芽黄素的含量

建立大鼠血浆中杨芽黄素的高效液相色谱串联质谱定量分析方法,考察大鼠灌胃给药后,杨芽黄素在其体内的药动学特征。血浆样品经乙腈试剂沉淀蛋白后,取上清液5 μL进样分析。珊瑚菜内酯作为内标物质,采用高效液相色谱串联质谱分析方法,检测大鼠血浆中杨芽黄素的浓度。选择色谱柱为Zorbax Eclipse Plus C₁₈柱(2.1 mm×50 mm, 1.8 μm),流动相为乙腈-0.1%甲酸水溶液(体积比60:40)梯度洗脱;采用电喷雾离子源,在正离子模式下的选择性反应离子监测m/z 268.8→225.9 (杨芽黄素)和m/z 300.9→233.0 (内标杨芽黄素)。杨芽黄素在0.5~200.0 ng/mL范围内线性关系良好;日间、日内精密度相对标准偏差为3.28%~13.12%,准确度相对误差-6.38%~7.60%;基质效应为98.43%~103.57%;提取回收率为93.36%~96.31%。经方法学完整验证,该分析方法可用于大鼠血浆中杨芽黄素的测定及其药动学评价研究。