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《中国兽医学报》2020,(7)
目前,病毒的研究多局限于细胞水平,精密组织切片(Precision-cut tissue slices,PCTS)与细胞水平相比不仅能够比较完整的保存正常组织结构、细胞间的联系及细胞极性,而且也能保证切片的生存能力,使其能够最大限度地模拟在体状态,为病毒的深入研究带来福音。本研究通过制备仔猪肺、肠的离体感染模型,并对离体模型的可行性进行验证,采用ATP检测试剂盒验证切片活性,并对其感染猪流行腹泻病毒(porcine epidemic diahorrea virus,PEDV) CV777和猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)GFP,进行RT-PCR检测和免疫荧光分析,结果显示小肠精密切片活性较好,维持在48 h左右,且能成功感染PEDV CV777和TGEV GFP,通过将肺脏气道内填充1.5%的低熔点琼脂糖,然后转移至切片机制作仔猪肺精密切片,观察气道上皮细胞纤毛活力,同时感染猪呼吸道冠状病毒(porcine respiratory coronavirus,PRCoV)后,进行RT-PCR检测和免疫荧光分析,结果显示肺精密切片生存力能够维持5 d左右,PRCoV成功感染肺精密切片且分布于气道上皮细胞周围,且病毒感染组纤毛活力比正常组下降快,结果表明,肺切片纤毛活性可作为病毒感染的重要评价指标。该研究可为模拟体内感染试验的离体模型提供理论依据,也为防制PEDV、TGEV、PRCoV提供新方法。 相似文献
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《中国预防兽医学报》2020,(8)
为研究猪繁殖与呼吸综合征病毒(PRRSV)在猪肺脏组织中的感染特性,本研究建立了高度分化的猪呼吸系统体外培养模型-猪肺组织精细切片(PCLS)。该培养体系包含肺脏组织中多种相关细胞并能够体现出肺脏组织的生理结构和生物学功能。本研究针对制备的猪PCLS进行生物学活性的鉴定,利用评估支气管上皮细胞纤毛摆动百分比的方法检测支气管上皮细胞纤毛活性,结果显示猪PCLS制备良好,在制备10 d以后仍保持95%以上的纤毛活性;利用活/死细胞染色法测定制备的猪PCLS体外培养后活细胞的比例,结果显示制备的猪PCLS在体外培养7 d后支气管上皮细胞和肺泡细胞仍为活细胞;利用间接免疫荧光试验测定猪PCLS中上皮细胞、杯状细胞和肺泡巨噬细胞(PAM)的完整性与分布情况,结果显示制备的猪PCLS上皮细胞和杯状细胞保存完整且猪PCLS中包含丰富的PAM。利用6株不同PRRSV分离株(HuN4、XD-15、WK-34、WK-38、LCL-75和DL-1510)以2.5×10~5TCID_(50)/片的剂量感染猪PCLS,检测不同PRRSV分离株在猪PCLS中的感染特性,结果表明不同PRRSV分离株在该培养体系中表现出不同的增殖能力。本研究表明猪PCLS可用于PRRSV的体外感染试验。本研究为PRRSV的体外研究提供了新的模型与方法,同时也为其它猪呼吸道病原体提供了新的研究平台。 相似文献
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正随着兽医临床诊断技术快速发展,动物传染病的诊断经常采用准确快速的PCR、荧光PCR等分子生物学方法、血清学检测方法等;但是,若想更直观详细地了解病理、确定病情发展进程,组织切片技术往往是必不可少的。根据不同的制作工艺,组织切片技术可分为石蜡切片和冰冻切;其中石蜡切片具有直观性、可保存时间长、成本低廉、运用范围广等优点,在疾病诊断、病理学研究等方面的运用尤其成熟,是兽医诊断中是最权威的诊断方法之一。因此,本文将对石蜡切片 相似文献
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柴芪颗粒抗菌及保肝作用研究 总被引：1，自引：1，他引：1
为探讨柴芪颗粒的保肝、体外抗菌作用和对小鼠免疫功能的影响,分别采用四氯化碳(CCl4)和D 氨基半乳糖(D GalN)诱导的小鼠肝损伤模型,给予不同剂量的柴芪颗粒(15、10、5 g/kg体重),测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的活性和肝组织谷胱甘肽(GSH)的含量。研究表明柴芪颗粒对四氯化碳(CCl4 )及D 氨基半乳糖(D GalN)所引起的小鼠肝损害有明显的保护作用,对血清ALT、AST活性升高有明显的降低作用(P<0.01),减少了CCl4 肝损伤小鼠戊巴比妥钠的睡眠时间;并能明显提高小鼠网状内皮系统的吞噬功能,体外试验有一定抑菌作用。可见柴芪颗粒具有抗菌、保肝和提高免疫功能的作用。 相似文献
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动态添加FSH对绵羊卵巢皮质组织体外培养的影响 总被引：1，自引：0，他引：1
本研究旨在探索促卵泡素(FSH)对体外培养绵羊卵巢原始卵泡和组织活性的影响。通过稳定或动态方式添加不同浓度FSH(1、10、50、100ng·mL-1),用组织学和免疫组化PCNA方法来评估FSH对体外培养绵羊卵巢皮质组织中原始卵泡激活、生长、存活以及颗粒细胞增殖活性的影响;此外通过测定培养液中雌激素含量,评估卵巢组织体外培养过程中活性的保持。结果表明,先升后降动态添加FSH对于卵泡发育、存活和生长以及颗粒细胞的增殖具有全面的促进作用;同时组织分泌雌激素能力也高于其他组。以稳定方式添加时,50ng·mL-1FSH在培养1d时显示对卵泡发育的显著促进作用(P0.05),添加10ng·mL-1以上浓度的FSH可以促进卵泡存活和生长(P0.05)。在整个培养期内,各培养组中雌激素产量持续增加,说明卵巢活性在本培养体系中可以得到很好的维持。本试验结果表明FSH可能参与早期卵泡的发生,动态添加FSH更有利于体外培养卵泡的发育和组织活性的维持。 相似文献
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Vietmeier J Niedorf F Bäumer W Martin C Deegen E Ohnesorge B Kietzmann M 《Veterinary research communications》2007,31(5):611-619
A study was performed to evaluate the use of precision-cut lung slices (PCLS) for studies on the contraction of equine airways.
Lungs of 10 horses were taken to prepare PCLS of ˜250 μm from equine lung tissue using a special microtome. The lung slices
were cultured and the enclosed small airways were monitored using a microscope with coupled digital camera, which was used
to determine the airway luminal area and diameter from digital images. As indicated by the beating of the ciliated epithelium
and reactivity of airways on methacholine challenge, the tissue slices were found to be viable for at least 24 h. The airways
were not precontracted, as indicated by a missing dilatory effect of 1 mmol/L clenbuterol. Bronchoconstriction induced by
both methacholine and histamine was found to be dose dependent. EC50 values based on luminal area were 1.12 μmol/L · /÷ 3.82 for methacholine and 0.68 μmol/L · /÷ 6.99 for histamine. In conclusion,
the PCLS technique is promising for studies on small airways in the equine lung. In the present study the basic principles
of in vitro (ex vivo) examinations with equine PCLS on airway reactivity were developed. 相似文献
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Green N Merton Boothe D Smith A Henderson R Whitley E 《Veterinary therapeutics : research in applied veterinary medicine》2010,11(2):E1-E11
Assay-based chemotherapeutic protocols are common in human gynecologic oncology, most notably for patients with ovarian or breast cancer. The current study examines ex vivo incubation conditions necessary for the assessment of sarcomatous tumor response to potential chemotherapeutic drugs. Slices of sarcomatous tumors were incubated in one of two culture media. Viability indices were measured and compared across time and between media. Neither medium was sufficient to support the growth of sarcomatous tumor tissue slices based on the indices studied. It is likely that sarcomatous tumors require a different approach for ex vivo assessment than their epithelial counterparts. Our long-term goal is to incubate tumor slices with chemotherapeutic agents to predict the in vivo tumor response based on the maintenance or loss of slice viability within this system. 相似文献
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饲用酶制剂在反刍动物营养中的应用进展 总被引：1，自引：0，他引：1
一般情况下,在瘤胃中粗饲料细胞壁组分的消化利用率较低,由于其复杂紧密的结构限制了动物对粗饲料的利用。因此,在反刍动物生产中提高粗饲料利用率的应用研究是非常必要的,不仅改善饲料利用率、提高动物生产性能,还为饲料资源的开发与利用乃至为畜牧业发展提供有效途径。而饲用酶制剂以其绿色、环保、安全等特点成为了该研究领域的热点。通常采用体外和半体内法评定酶制剂处理对粗饲料体外发酵和瘤胃降解特性的影响,并采用体内法评价酶制剂在反刍动物营养中的应用效果。研究表明,外源酶制剂的应用能够有效提高饲料消化率、奶牛产量、肉牛增重和肉羊增重,但其效果不一致,其效果受很多因素的影响,包括酶种类、酶活性、饲粮组成、添加水平、添加方式、动物种类及生长性能等。另外,由于人们对所用酶产品的生化特性掌握不足,导致了对其作用机理、优化条件及影响其作用效果因子的了解不够充分。如酶活性作为酶制剂最主要的生化特性,在使用前往往被忽略检测,并且目前国内外对酶活性的检测及质量评定还没有统一的标准。然而研究结果间直接进行比较也是不科学不合理的。本研究主要从饲用酶制剂的发展历程,反刍动物营养中纤维降解酶的作用机理、应用效果和影响其作用效果的因素以及存在问题等方面进行了综述,旨在为饲用酶制剂在反刍动物营养中的应用提供系统的科学知识。 相似文献
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Cuniberti B Odore R Barbero R Cagnardi P Badino P Girardi C Re G 《Veterinary journal (London, England : 1997)》2012,191(3):327-333
Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX), and the inhibition of COX-2 rather than COX-1 can limit the onset of NSAID-related adverse effects. The pharmacodynamic properties of eltenac, naproxen, tepoxalin, SC-560 and NS 398 in healthy horses were investigated using an in vitro whole blood assay. To predict COX selectivity in clinical use, eltenac and naproxen were also studied ex vivo after intravenous administration. SC-560 acted as a selective COX-1 inhibitor, tepoxalin as a dual inhibitor with potent activity against COX-1, and NS 398 as a preferential COX-2 inhibitor. Eltenac was a preferential COX-2 inhibitor in vitro but un-selective in the ex vivo study. Naproxen maintained its non-selectivity both in vitro and ex vivo. These findings have demonstrated that in vitro studies may not accurately predict in vivo NSAID selectivity for COX and should be confirmed using an ex vivo whole blood assay. 相似文献
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Glycerolipid biosynthesis by porcine adipose tissue homogenates did not yield the 90+% triacylglycerol observed in situ. Consequently, we compared intact tissue slices and various subcellular fractions to characterize the usefulness of such systems to assess glycerolipid biosynthesis in vitro. Glycerolipid biosynthesis by porcine adipose tissue homogenates was measured in vitro using either [14C]-fatty acid or [14C]-glycerol-3-phosphate (G3P) as a radiolabelled substrate. Removal of residual 14C-labelled fatty acid from lipid extracts was difficult. Because G3P is soluble in water, residual [14C]-G3P separated easily from the glycerolipid-containing organic phase and, thus, was the preferred radiolabelled substrate. With tissue slices, glycerol and G3P were minimally incorporated into lipid so that [14C]-fatty acid was the preferred radiolabelled tracer. A washing procedure followed by thin layer chromatography was devised to separate residual [14C]-fatty acid from glycerolipids, including phospholipids. Fatty acid esterification into glycerolipids in tissue slices yielded about 4% phospholipids, whereas with homogenates, esterification yielded up to 50% phospholipids. Comparison of several subcellular fractions indicated that microsomes contained most of the glycerolipid biosynthetic activity when activity was expressed on a protein basis. However, when activities were expressed on a tissue wet weight basis, the 700 x g infranate and the 10,000 x g supernate had about equal activity that was far greater than the microsomes. The 700 x g infranate was the preferred enzyme preparation for assay of the entrance of G3P into the pathway as well as the capacity to synthesize triacylglycerol. Several methods of freezing and storing tissue or 700 x g infranates were not acceptable. Freezing of the 700 x g infranate in liquid N2 with storage at -80 degrees C may be an acceptable procedure. 相似文献
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Stegemann MR Sherington J Coati N Brown SA Blanchflower S 《Journal of veterinary pharmacology and therapeutics》2006,29(6):513-524
The pharmacokinetics of the novel cephalosporin cefovecin were investigated in a series of in vivo, ex vivo and in vitro studies following administration to adult cats at 8 mg/kg bodyweight. Bioavailability and pharmacokinetic parameters were determined in a cross-over study after intravenous (i.v.) and subcutaneous (s.c.) injections. [14C]cefovecin was used to evaluate excretion for 21 days after s.c. administration. Protein binding was determined in vitro in feline plasma and ex vivo in transudate from cats surgically implanted with tissue chambers. After s.c. administration, cefovecin was characterized by rapid absorption with mean peak plasma concentrations of 141+/-12 microg/mL being achieved within 2 h of s.c. injection with full bioavailability (99%). The mean elimination half-life was 166+/-18 h. After i.v. administration, volume of distribution was 0.09+/-0.01 L/kg and mean plasma clearance was 0.35+/-0.04 mL/h/kg. Approximately 50% of the administered radiolabelled dose was eliminated over the 21-day postdose period via urinary excretion and up to approximately 25% in faeces. In vitro and ex vivo plasma protein binding ranged from 99.8% to 99.5% over the plasma concentration range 10-100 microg/mL. Ex vivo protein binding in transudate was as low as 90.7%. From 8 h postdose, concentrations of unbound (free) cefovecin in transudate were consistently higher than in plasma, with mean unbound cefovecin concentrations being maintained above 0.06 microg/mL (MIC90 of Pasteurella multocida) in transudate for at least 14 days postdose. The slow elimination and long-lasting free concentrations in extracellular fluid are desirable pharmacokinetic attributes for an antimicrobial with a 14-day dosing interval. 相似文献
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Functional characterization of equine dendritic cells propagated ex vivo using recombinant human GM-CSF and recombinant equine IL-4 总被引：3，自引：0，他引：3
Naive T cells can be activated both in vivo and in vitro by specialized antigen presenting cells, dendritic cells (DC), with potent antigen-specific, immunostimulatory activity. Indeed, DC can provide an extremely powerful and important immunological tool by which to potentiate the immune response for specific recognition of foreign antigens. Until recently, the direct isolation of DC from PBMC required laborious procedures with extremely poor yields (<0.1%). Methods have been developed for the human, lower primate, and murine model systems to propagate large numbers of DC from PBMC or bone marrow ex vivo with various cytokines. However, all other model systems, including equine, still require the laborious isolation procedures to obtain DC. In this study, we have adapted the methods developed for the human system to generate large numbers of equine DC from PBMC precursors using recombinant human GM-CSF and recombinant equine IL-4. Our report is the first documentation of ex vivo generated DC from PBMC in a domesticated animal model system. Equine DC derived from PBMC were rigorously characterized by analyzing morphological, phenotypic, and functional properties and were determined to have similar attributes as DC generated from human PBMC. Equine DC appeared stellate with large projectiles and veils and had cell surface antigens at similar levels as those defined on human and murine DC. Furthermore, functional attributes of the DC included rapidly capturing antigens by pinocytosis, receptor-mediated endocytosis, and phagocytosis, activating naive T cells in a mixed leukocyte reaction to a much greater extent than macrophage or lymphoblasts, presenting soluble and particulate antigen 10-100 fold more effectively to T cells on a per cell basis than macrophage or lymphoblasts, and presenting soluble and particulate antigen to both CD4+ and CD8+ T cells. Taken together, our study provides a framework by which equine DC can now be readily produced from PBMC precursors and presents an impetus for and model by which DC can be simply generated in other animal model systems. 相似文献
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The pharmacokinetics and pharmacodynamics of danofloxacin were studied in calves after intravenous (IV) and intramuscular (IM) administration, at a dose of 1.25 mg/kg in a two period cross-over study, using tissue cages to monitor aspects of extravascular distribution. Danofloxacin had a high volume of distribution (3.90 L/kg) and relatively rapid clearance (1.02 L/kgh) after IV dosing. Terminal half-life was 2.65 and 4.03 h, respectively, after IV and IM administration. Danofloxacin penetrated slowly into and was cleared slowly from tissue cage fluid (transudate), elimination half-life (10.2 h after IV and 8.9 h after IM dosing) being greater than for serum. The antibacterial actions of danofloxacin against the pathogen Mannheimia haemolytica 3575 were established in vitro in Mueller Hinton Broth, serum and transudate. These data were used together with in vivo pharmacokinetic parameters, C(max) and AUC to determine the surrogate markers of antimicrobial activity, C(max)/MIC, AUC/MIC and T>MIC.The antibacterial actions of danofloxacin were also determined ex vivo in serum and transudate samples harvested at pre-determined times after IM danofloxacin dosing. Ex vivo AUC/MIC data were integrated with ex vivo bacterial count to establish values producing a bacteriostatic action, inhibition of bacterial count by 50%, reduction in bacterial count by 99.9% (bactericidal action) and elimination of bacteria. Mean values were, respectively, 15.9, 16.7, 18.15 and 33.5h for serum and 15.0, 16.34, 17.8 and 30.7 h for transudate. The AUC/MIC-effect relationships for serum may be regarded as representative of a shallow compartment of blood and well perfused tissues, whilst AUC/MIC-effect relationships for transudate may be considered to represent a deep peripheral compartment of poorly perfused tissues. A novel approach to selecting antimicrobial drug dosage for evaluation in clinical trials, using AUC/MIC values producing either bactericidal activity or elimination of bacteria together with MIC(90) values for calf pathogens, is proposed. This approach can be expected to optimise efficacy and minimise the development of resistance. 相似文献
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Ryan PL Christiansen DL Hopper RM Walters FK Moulton K Curbelo J Greene JM Willard ST 《Journal of animal science》2011,89(5):1541-1551
Uterine and placental infections are the leading cause of abortion, stillbirth, and preterm delivery in the mare. Whereas uterine and placental infections in women have been studied extensively, a comprehensive examination of the pathogenic processes leading to this unsatisfactory pregnancy outcome in the mare has yet to be completed. Most information in the literature relating to late-term pregnancy loss in mares is based on retrospective studies of clinical cases submitted for necropsy. Here we report the development and application of a novel approach, whereby transgenically modified bacteria transformed with lux genes of Xenorhabdus luminescens or Photorhabdus luminescens origin and biophotonic imaging are utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology uses highly sensitive bioluminescence imaging camera systems to localize and monitor pathogen progression during tissue invasion by measuring the bioluminescent signatures emitted by the lux-modified pathogens. This method has an important advantage in that it allows for the potential tracking of pathogens in vivo in real time and over time, which was hitherto impossible. Although the application of this technology in domestic animals is in its infancy, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary, and gastrointestinal systems as primary tissues for pathogen invasion after experimental infection of pregnant mares with lux-modified Escherichia coli. It is important that pathogens were not detected in other vital organs, such as the liver, brain, and cardiac system. Such precision in localizing sites of pathogen invasion provides potential application for this novel approach in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species. 相似文献
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Aliabadi FS Ali BH Landoni MF Lees P 《Veterinary journal (London, England : 1997)》2003,165(2):104-118
The pharmacokinetics and pharmacodynamics of danofloxacin were studied in the camel in a two period cross-over study. After intravenous (i.v.) administration at a dose rate of 1.25 mg/kg, the pharmacokinetics of danofloxacin indicated a high volume of distribution (V(d(area))=3.43 L/kg), relatively rapid clearance (0.44 L/kg/h) and half-life of 5.37 h. After intramuscular (i.m.) dosing absorption was complete (F=114.5) and rapid (T((1/2)abs)=0.12 h) and terminal half-life was 5.71 h. Danofloxacin penetrated fairly slowly into both inflamed (exudate) and non-inflamed (transudate) tissue cage fluids and was cleared slowly from these fluids, elimination half-life being at least twice that for serum for both exudate and transudate after both i.v. and i.m. dosing. The antibacterial actions of danofloxacin against the camel pathogen Escherichia coli 0157-H7 were determined by measurement of minimum inhibitory concentration (MIC) in vitro (single measurement) and ex vivo measurements of bacterial count at nine times between one and 48 h after i.m. dosing in each of the fluids, serum, exudate, and transudate. Using in vitro MIC data and in vivo pharmacokinetic parameters, the surrogate markers of antimicrobial activity, C(max)/MIC, AUC/MIC and T>MIC, were determined for all three fluids. The ex vivo serum AUC(24 h)/MIC data were integrated with reduction in bacterial count to provide values producing a bacteriostatic action (no change in bacterial count), inhibition of bacterial count by 50%, reduction in bacterial count by 99.9% (bactericidal action) and elimination of bacteria. Mean AUC(24h)/MIC values were 17.20, 20.07, 21.24, and 68.37 h, respectively. To describe the latter, the introduction of a new term to supplement MIC and minimum bactericidal concentration (MBC) is proposed, namely minimum elimination concentration (MEC). A novel means of designing antimicrobial drug dosage schedules for evaluation in clinical trials is proposed, using ex vivo AUC(24h)/MIC values for bactericidal activity and elimination of bacteria together with MIC(90) data for camel pathogens. 相似文献