应用噻唑蓝法初步研究兔外周血单个核细胞转化的条件

应用噻唑蓝法研究了ＰＨＡ－Ｐ引起兔外周血单个核细胞（ＰＢＭＣ）转化的最适条件,培养时间为４８ｈ,ＰＢＭＣ密度为５×１０１２／Ｌ,ＰＨＡ－Ｐ的终浓度为０．５ｍｇ％。并测定了在此最适条件下的吸光值（ＯＤ）,为０．２７６±０．１４２。     

Identification of bovine dendritic cell phenotype from bovine peripheral blood

Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.     

猪外周血单个核细胞cDNA酵母表达文库的构建及与猪瘟病毒E2蛋白相互作用细胞蛋白的筛选

为研究猪瘟病-毒(CSFV)蛋白与宿主细胞蛋白之间的相互作用,本研究通过SMART技术合成猪外周血单个核细胞(PBMC)双链cDNA,以携带与载体pGADT7-Rec重组位点同源序列的特异引物经long-distance PCR扩增双链cDNA.纯化双链cDNA并与载体pGADT7-Rec共转化酵母菌株Y187,经同源重组构建PBMC cDNA酵母表达文库.以CSFV E2蛋白为诱饵进行酵母双杂交筛选,得到阳性克隆根据序列比对分析结果,进一步进行共转化验证.结果显示筛选到17个与CSFV E2蛋白相互作用的宿主细胞蛋白,基因注释(GO)分析表明这些蛋白分别参与免疫、代谢、细胞生长与增殖、生物调节等过程,为研究它们与CSFV E2的相互作用奠定了基础.     

Culture and Stimulation of Tammar Wallaby Lymphocytes

We describe the culture and stimulation of lymphocytes from the model marsupial, the tammar wallaby (Macropus eugenii). We also describe the capacity of tammar wallaby lymphocytes isolated from blood, spleen and lymph nodes to produce soluble immunomodulatory factors. Culture conditions were optimized for mitogen-driven stimulation using the plant lectin phytohaemagglutinin (PHA). Products secreted by stimulated cells were harvested and crudely fractionated before they were added back to freshly isolated lymphocytes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, both stimulatory and inhibitory bioactive factors were detected in serum-free supernatants harvested from mitogen-treated peripheral blood mononuclear cells. This paper describes the capacity of leukocytes of the tammar wallaby to respond to mitogenic stimulation and to produce soluble, low-molecular-weight bioactive molecules that possess cytokine-like activity.     

CD58对猪PBMC转化的作用

应用ＭＴＴ比色法,测定了由ＳＲＢＣ膜及ＰＲＢＣ膜提取的ＣＤ５８对猪ＰＢＭＣ转化的影响。结果证明,两种ＣＤ５８成分均能显著提高ＰＨＡ－Ｐ对ＰＢＭＣ的激活作用（Ｐ＜０．０１或Ｐ＜０．０５）,单独应用也表现出对ＰＢＭＣ的活化效应,且上述作用具有剂量依赖性。ＣＤ５８浓度达２．５ｇ·Ｌ－１时作用最强（Ｐ＜０．０５）,超过该浓度则活化作用减弱,其作用的规律与ＲＢＣ一致。比较分析表明,猪源ＣＤ５８在最佳剂量时,单独刺激作用或对ＰＨＡ－Ｐ诱导的ＰＢＭＣ转化的协同作用均显著大于绵羊源ＣＤ５８（Ｐ＜０．０５）,ＰＲＢＣ的协同刺激作用也大于ＳＲＢＣ．     

猪外周血单个核细胞增殖反应影响条件的探讨

应用 Ｌ１６ （４５） 正交试验, 对影响猪 Ｐ Ｂ Ｍ Ｃ 增殖反应的几个因素, 包括培养时间、 Ｐ Ｈ Ａ 浓度、细胞浓度等三个因素四个水平进行了比较和探索。试验表明几个因素对 Ｐ Ｂ Ｍ Ｃ 的增殖反应均有显著的影响（ Ｐ＜ ００５） , Ｐ Ｂ Ｍ Ｃ 增殖反应的 Ｍ Ｔ Ｔ 比色法的最佳反应条件组合为 Ｐ Ｂ Ｍ Ｃ 浓度为１ ×１０６／ｍｌ, 植物血凝素 Ｐ Ｈ Ａ 的浓度为１２５μｇ／ ｍｌ, 细胞培养时间为２４ 小时; 影响增殖反应的先后顺序为 Ｐ Ｂ Ｍ Ｃ 浓度、 Ｐ Ｈ Ａｐ 浓度及细胞培养时间。同时确定了最有利于 Ｐ Ｂ Ｍ Ｃ 增殖的犊牛血清浓度为１０ ％ 。     

All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.     

猪流感病毒毒株感染对猪外周血淋巴细胞炎性因子转录时相的影响

试验用猪流感病毒（Swine influenza virus,SIV）（毒株）体外感染猪外周血淋巴细胞（Peripheral blood mononuclear cell,PBMC）,利用real—timePCR方法对病毒感染不同时间细胞中的病毒量和炎性细胞因子mRNA表达量进行检测。结果显示,感染后1h就可检测到病毒,但病毒量相对较小;在24h时病毒开始大量增殖;在72h病毒量达到最高峰,为1h时20倍以上。IL-1βmRNA的表达量在48h增加到最高;IL-6mRNA的表达量在感染后1h内显著增加;IL-8mRNA的表达量在12h时达到最大量;IL-12P35mRNA的表达量在72h迅速上升,为对照的56．1倍;IL-12P40mRNA的表达量在1～2h表达量较高;IL-13在72h达到最大值,为对照的30．7倍;IL-17mR—NA的表达量在72h达到最大值,为对照的4．9倍;IL-18mRNA的表达量在12h达到对照的1．9倍。结果表明,SIV感染PBMC后,随着病毒的大量复制,多种炎性细胞因子表达量显著增加。     

Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains

cDNAs encoding four different canine immunoglobulin G (caIgG) γ chains were identified in this study. One of these IgG γ chain cDNAs, (caIgG-A), represents 92.5% of the IgG γ chain cDNAs in a dog spleen cell cDNA library; a second partial IgG γ chain cDNA (caIgG-B) was also identified in the library. The other two IgG γ chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG γ chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG γ chains, the hinge regions were distinct, sharing only 19.0–35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG γ chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy γ chains (A, B, C and D), respectively. Specific primers were designed based on caIgG γ chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG γ chain mRNA levels in canine PBMC samples.     

Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on the infecting bacterial genotype

In this study we investigated the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows. We tested the hypotheses that infection status of cows affects macrophage killing ability and that survival of M. paratuberculosis in macrophages is dependent on the strain. Peripheral blood mononuclear cells (PBMC) were obtained from Johne's disease-positive (n = 3) and age and stage of lactation matched Johne's disease-negative (n = 3) multiparious cows. Following differentiation, MDMs were challenged in vitro with four M. paratuberculosis strains of different host specificity (cattle and sheep). Two hours and 2, 4, and 7 days after infection, ingestion, and intracellular survival of M. paratuberculosis strains were determined by fluorescence microscopy. There was no effect of the origin of MDMs (Johne's disease-positive or control animals) on phagocytosis, survival of bacteria, or macrophage survival. In contrast, important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which may make it important to include strain typing when designing control programs.