天门冬多糖对小鼠脾脏淋巴细胞体外增殖的影响

本试验旨在研究天门冬多糖对小鼠脾脏淋巴细胞体外增殖的影响。从正常小鼠分离脾脏淋巴细胞体外培养,在细胞培养体系中分别加入终浓度为50、100、200、400 mg/L天门冬多糖或配合刀豆素(5 mg/L)作用24、487、2 h,以MTT法测定细胞的增殖程度。结果表明,天门冬多糖可显著促进体外培养的小鼠脾脏淋巴细胞增殖,并与ConA有协同作用。     

分级醇沉刺五加多糖不同浓度对小鼠脾细胞增殖和IL-2表达的影响

为了研究不同醇沉浓度刺五加多糖对脾细胞增殖和细胞因子IL-2表达的影响,试验采用分级醇沉的方法对刺五加多糖进行醇沉得到粗糖,并去蛋白得到精制糖,研究分级醇沉刺五加多糖不同浓度对小鼠脾细胞增殖、ConA和LPS诱导的脾淋巴细胞的增殖反应和脾细胞产生细胞因子表达的影响。结果表明:40%醇沉组多糖浓度为7.8～125μg/mL时均能促进脾细胞增殖,并且其促进脾细胞增殖作用与多糖浓度呈剂量依赖性,促增殖作用增强;40%醇沉组多糖浓度为15.62～125μg/mL时能促进ConA诱导的脾淋巴细胞的增殖反应;各醇沉组多糖浓度为15.62～125μg/mL时均能显著促进LPS诱导的脾淋巴细胞的增殖反应;20%、40%、60%醇沉组多糖浓度为3.9～7.81μg/mL时IL-2的表达水平呈上升趋势;并且随药物浓度的增大细胞因子表达量先增多后减少。说明以40%醇沉刺五加多糖免疫增强效果优于其他醇沉多糖。     

蕨麻多糖对小鼠淋巴细胞增殖和一氧化氮分泌的影响

为探讨蕨麻多糖（Potentilla anserine polysaccharide，PAP）免疫调节的作用机理，观察了PAP对小鼠脾淋巴细胞体外增殖和分泌一氧化氮的影响。结果显示，50mg／L PAP配合ConA或LPS（lipopolysaccharide，脂多糖）能使小鼠脾淋巴细胞在体外显著增殖（P〈0．05）；100、200及400mg／L PAP配合ConA或LPS可使小鼠脾淋巴细胞在体外极显著增殖（P〈0．01）；与对照组相比，PAP以不同浓度（50～400mg／L）处理小鼠脾淋巴细胞后一氧化氮分泌量均显著升高（P〈0．05）。试验结果表明，蕨麻多糖能明显促进体外培养的小鼠脾淋巴细胞增殖和分泌一氧化氮，与ConA或LPS有协同作用。     

九种中药成分对体外培养小鼠淋巴细胞功能的影响

为进一步探索黄芪多糖等9种中药成分免疫增强活性的作用机理并比较各成分之间免疫增强活性的强弱，在体内试验的基础上，用脾淋巴细胞增殖反应和抗体夹心酶联免疫吸附试验测定了它们对体外培养的小鼠淋巴细胞功能的影响。结果表明，人参皂甙、蜂胶黄酮、黄芪多糖、淫羊藿黄酮、淫羊藿多糖都能显著地直接或协同ConA刺激脾和外周血淋巴细胞增殖，也能显著增加LPS诱导的脾淋巴细胞增殖活性和显著升高体外培养的小鼠脾淋巴细胞产生的IgG水平；当归多糖能协同ConA和LPS的诱导活性，提高小鼠脾淋巴细胞体外培养系统的IgG水平；黄芪皂甙能直接和协同LPS刺激淋巴细胞增殖，板蓝根多糖则仅能促进LPS的诱导活性，蜂胶多糖的作用则均不明显。     

4种中药多糖对小鼠脾淋巴细胞增殖的影响

本试验旨在研究黄芪多糖(APS)、芦荟多糖(AP)、银耳多糖(TFPS)、黑木耳多糖(AAP)4种中药多糖对小鼠脾淋巴细胞增殖的影响.将4种多糖体外作用于小鼠脾淋巴细胞;采用MTT法测细胞增殖活性.结果显示,与对照组相比较,4种多糖在浓度为400 μg/mL时均能促进静止态淋巴细胞增殖;多糖协同ConA或LPS作用于淋巴细胞,4种多糖表现不同程度的增殖促进作用.在静止态淋巴细胞增殖以及协同丝裂原作用于淋巴细胞的增殖试验中,芦荟多糖所表现的促进增殖作用较强于其他3种多糖.     

蛹虫草多糖对鸡脾脏淋巴细胞的影响

试验将7种浓度蛹虫草多糖(CMP)单独或与刀豆蛋白A(ConA)、脂多糖(LPS)同时加入鸡脾脏淋巴细胞的培养体系中,培养48 h时用MTT法测定淋巴细胞增殖(A570值)的变化;将5种浓度的CMP 40%乙醇提取物(CMP40)、CMP 50%乙醇提取物(CMP50)分别与ConA加入到鸡脾淋巴细胞的培养体系中,培养24 h后收集细胞培养上清液,测定白细胞介素-2(IL-2)和γ-干扰素(IFN-γ)的含量。结果显示,CMP在31.25~2 000μg/mL时,CMP40+ConA组的A570值显著高于ConA对照组;CMP在31.25~500μg/mL时,CMP50+ConA组A570值显著高于ConA对照组,CMP40+LPS组、CMP50+LPS组的A570值显著高于LPS对照组;CMP40在125~1 000μg/mL时、CMP50在500μg/mL时,各多糖组的A570值显著高于细胞对照组;CMP40和CMP50在250~1 000μg/mL时,各多糖组的IL-2和IFN-γ浓度显著高于对照组。结果表明,CMP40和CMP50在合适的剂量能单独或协同ConA、LPS刺激鸡的T、B淋巴细胞增殖,促进淋巴细胞分泌IL-2和IFN-γ,从而增强鸡的细胞免疫能力。     

白花蛇舌草多糖体外免疫活性研究

目的:探讨白花蛇舌草多糖对小鼠胸腺和脾脏体外免疫功能的影响。方法:采用MTT法研究不同浓度(500μg/m L、250μg/m L、200μg/m L、150μg/m L、100μg/m L、50μg/m L、25μg/m L)的白花蛇舌草多糖对体外培养的小鼠胸腺和脾脏淋巴细胞的代谢及其与Con A或LPS协同诱导的转化作用、小鼠胸腺和脾脏巨噬细胞吞噬活性及自然杀伤细胞活性的影响。结果:白花蛇舌草多糖可显著提高脾脏巨噬细胞吞噬功能(P0.01),促进脾脏淋巴细胞代谢及其与Con A或LPS协同诱导的转化作用(P0.01),但对小鼠胸腺淋巴细胞代谢及其与Con A或LPS协同诱导的转化作用、小鼠胸腺巨噬细胞吞噬功能无显著性影响(P0.05);白花蛇舌草多糖能够显著增强小鼠胸腺和脾脏NK细胞活性(P0.01),并呈现明显的量效双向作用关系。结论:一定浓度范围内的白花蛇舌草多糖可提高小鼠脾脏和胸腺淋巴细胞、巨噬细胞和NK细胞的体外免疫活性。     

淫羊藿多糖对鸡脾脏淋巴细胞的调节作用研究

为研究淫羊藿多糖(EPS)体外增强免疫活性,采用酶学检测法测定淫羊藿多糖活性及对鸡脾脏淋巴细胞的增殖作用。根据活性检测结果,选择用50%的乙醇醇沉的淫羊藿多糖进行安全浓度试验;根据安全浓度检测结果,选择2 500.00μg/mL、1 250.00μg/mL、625.00μg/mL、312.50μg/mL、156.25μg/mL 5个浓度,用MTT法测定淫羊藿多糖单独和协同LPS刺激对鸡脾脏淋巴细胞增殖率的影响。结果显示:不同浓度乙醇醇沉的淫羊藿多糖均具有良好的活性,但乙醇浓度为50%醇沉的淫羊藿多糖活性最佳。淫羊藿多糖对鸡脾脏淋巴细胞的安全浓度为2 500.00μg/mL。淫羊藿多糖无论是单独还是协同LPS刺激脾脏淋巴细胞,均显示出不同程度增强脾脏淋巴细胞的活性,且均在浓度为1 250.00μg/mL时显示出较强的体外增强鸡脾脏淋巴细胞增殖的能力,说明淫羊藿多糖在安全浓度范围内可刺激鸡脾脏淋巴细胞增殖,从而实现调节免疫功能的作用。     

猴头菇多糖对小鼠脾淋巴细胞体外NO分泌及iNOS mRNA表达的影响

为了研究猴头菇多糖(Hericium erinaceus polysaccharide,HEP)对小鼠脾淋巴细胞NO生成及iNOS mRNA表达的影响。采用Griess法测定HEP单独或协同ConA诱导脾淋巴细胞分泌NO含量;RT-PCR法检测脾淋巴细胞iNOS mRNA表达量。结果与细胞对照组比较,HEP在25~400μg/m L的浓度范围内能单独诱导脾淋巴细胞NO分泌及iNOS mRNA表达(P0.05);与ConA对照组比较,HEP在100~400μg/m L浓度范围内能协同Con A诱导脾淋巴细胞NO分泌及iNOS mRNA表达(P0.05)。表明HEP能单独或协同Con A诱导小鼠脾淋巴细胞分泌NO,此作用可能是通过促进i NOS mRNA表达而实现。     

银杏叶复方对脾虚小鼠小肠淋巴细胞体外增殖的影响

为了探究不同浓度的银杏叶复方在脂多糖(LPS)或植物血凝集素(PHA)刺激下对脾虚小鼠小肠淋巴细胞体外增殖的影响,试验用不同浓度的银杏叶复方(200μg/mL、100μg/mL、10μg/mL、1μg/mL、0.1μg/mL)同LPS或PHA刺激小肠淋巴细胞分裂增殖的方法研究对小鼠淋巴细胞体外增殖的影响。结果表明:对于脾虚小鼠小肠B淋巴细胞,以200μg/mL、100μg/mL的浓度刺激效果最佳(P0.05);相比于1μg/mL、0.1μg/mL浓度,浓度为200μg/mL时更有利于正常小鼠小肠B淋巴细胞增殖(P0.05);对于脾虚小鼠小肠T淋巴细胞,以100μg/mL浓度最佳(P0.05),浓度为200μg/mL和100μg/mL时对正常小鼠小肠T淋巴细胞的刺激增殖效果显著(P0.05)。说明银杏叶复方浓度为200μg/mL、100μg/mL时能有效刺激正常和脾虚小鼠的小肠淋巴细胞分裂增殖。     

胸腺素α_1对鸡脾脏淋巴细胞增殖转化及IL-2基因表达与分泌的影响

为进一步阐明胸腺素α1(Tα1)的作用机制,采用MTT比色法、RT-PCR相对半定量法和RIA法研究了不同浓度的Tα1对离体培养的鸡脾脏淋巴细胞增殖转化、IL-2基因表达及其分泌的影响。结果表明,10μg/mL和1μg/mL Tα1处理鸡脾脏淋巴细胞能显著提高由ConA诱导的淋巴细胞增殖转化率(P<0.05),显著提高鸡脾脏淋巴细胞IL-2基因表达水平以及细胞培养上清液中的IL-2含量(P<0.05),且以10μg/mL浓度的效果最佳。0.1μg/mL的Tα1对脾脏淋巴细胞增殖转化和IL-2基因表达与分泌无显著作用(P>0.05)。提示,Tα1对鸡免疫功能的调节同其剂量有关,并可能在基因转录水平上调节IL-2的分泌。     

胸腺素α_1调节鸡脾脏淋巴细胞免疫功能的信号转导途径

在无菌条件下,从健康墟岗黄鸡体内取出脾脏,分离淋巴细胞并进行体外培养。在细胞培养液中分别加入终浓度为0.01 mol/L咖啡碱、0.05 mol/L EDTA、0.01 mol/L氯化锂和5μg/mL甲派氟丙嗪,然后用10μg/mL胸腺素α1(Tα1)处理,再加入20μg/mL ConA,研究Ca2+-肌醇磷脂-蛋白激酶系统和cAMP-肌醇磷脂-蛋白激酶系统在Tα1调节淋巴细胞免疫功能中的作用,揭示Tα1调节免疫功能的信号转导机制。结果表明,咖啡碱处理能显著增强Tα1促进脾脏淋巴细胞增殖转化、IL-2的基因表达,提高培养液中IL-2浓度(P<0.05);而EDTA处理能显著抑制Tα1促进脾脏淋巴细胞增殖转化、IL-2的基因表达与分泌的作用(P<0.05);氯化锂和甲派氟丙嗪对Tα1的免疫调节作用的影响不明显(P>0.05)。结果提示,Tα1调节鸡脾脏淋巴细胞的增殖和IL-2的基因表达与分泌的作用,主要通过cAMP、Ca2+和PKC等第二信使的介导。     

猪外周血T淋巴细胞增殖反应MTT检测方法的建立

T细胞增殖反应是宿主T细胞识别病原的结果,也是宿主细胞免疫应答的重要指标之一。为了便于检测猪群在病原感染或者疫苗免疫过程中产生的细胞免疫应答,本研究应用MTT法建立了体外检测猪外周血T细胞增殖反应的研究方法。通过密度梯度离心法从外周血分离得到外周血单个核细胞（PBMC）,然后利用单核细胞和淋巴细胞不同的生长特性（贴壁与否）,弃掉贴壁的单核细胞,获得外周血淋巴细胞(PBL)。外周血淋巴细胞的流式分析结果显示,分离获得的PBL中T细胞所占比例达到了80%以上。应用MTT法分析了非特异性刺激物刀豆蛋白A（ConA）的浓度和细胞培养密度对T细胞增殖的影响。结果显示,ConA的工作浓度为5 μg/mL、细胞培养密度为2×106/mL时T细胞的增殖反应最强烈。本研究所建立的猪外周血T细胞增殖反应检测法可以为研究猪针对病原或疫苗的细胞免疫反应提供参考。     

重组红火蚁毒素蛋白SoliⅣ致病机理的初步研究

作者通过研究重组红火蚁毒素蛋白Solenopsis invictaⅣ (SoliⅣ)对兔及外周血淋巴细胞的影响,探讨红火蚁毒素蛋白致病机制。通过分离兔外周血淋巴细胞进行培养,经不同浓度的重组红火蚁毒素蛋白SoliⅣ分别和细菌脂多糖(LPS)、刀豆蛋白(ConA)共同刺激后,MTT法测定淋巴细胞的增殖情况;体内试验：用重组蛋白SoliⅣ从兔背部皮下注入,观察兔的临床反应,定期采血,用ELISA方法测定兔血清中白介素 4（IL-4）和总IgE的变化。重组红火蚁毒素蛋白SoliⅣ浓度为25、50、75 μg/ml和LPS共刺激时,与单独LPS刺激对照组相比,淋巴细胞增殖活性显著增高（P<0.05）;浓度为15、25、50、75 μg/ml和ConA共刺激时,与单独ConA刺激对照组比较,淋巴细胞增殖活性显著增高（P<0.05）;重组SoliⅣ过敏兔的血清中IL-4和总IgE的水平升高,在20 h左右达到最高值。试验结果表明,一定浓度的重组红火蚁毒素蛋白SoliⅣ能引起体外培养的T、B淋巴细胞增殖,过敏体质兔在重组红火蚁毒素蛋白SoliⅣ刺激后能引起Ⅰ型变态反应。     

脱氧雪腐镰刀菌烯醇与玉米赤霉烯酮联合暴露对体外培养鸡脾脏淋巴细胞内环境稳态的影响

本试验旨在研究脱氧雪腐镰刀菌烯醇(DON)与玉米赤霉烯酮与(ZEA)联合暴露对体外培养鸡脾脏淋巴细胞内环境稳态的影响。分别以0.012 50μg/mL DON+0.006 25μg/mL ZEA、0.050μg/mL DON+0.025μg/mL ZEA、0.2μg/mL DON+0.1μg/mL ZEA、0.8μg/mL DON+0.4μg/mL ZEA对体外培养鸡脾脏淋巴细胞进行联合暴露培养,48 h后测定细胞膜ATP酶(Ca~(2+)-ATP酶、Na~+/K~+-ATP酶)活性以及细胞内pH、Ca~(2+)水平和钙调蛋白(CaM)的mRNA表达水平。同时设不添加毒素的空白对照组。结果表明:添加毒素的各试验组间,细胞内Ca~(2+)水平、CaM mRNA表达水平随毒素浓度的升高而增加,且添加毒素的各试验组均显著或极显著高于空白对照组(P0.05或P0.01)。细胞内pH以及细胞膜Ca~(2+)-ATP酶与Na~+/K~+-ATP酶活性均随毒素浓度的升高而降低,且添加毒素的各试验组均显著或极显著低于空白对照组(P0.05或P0.01)。由此得出,DON、ZEA联合暴露导致体外培养鸡脾脏淋巴细胞内酸化、离子平衡失调等一系列细胞内环境稳态失衡,且呈剂量依赖性。     

Lymphocyte transformation test in veterinary clinical immunology

Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavlin A (Con A), pokeweed mitogen (PWM), and pl ytohemagglutinin (PHA) at optimal concentrations (Con A, 15 μg/ml, PWM and PHA, 5 μg/ml) and decreased concentrations (Con A, 5 μg/ml, PWM and PHA, 1 μg/ml). Mitogenesis induced by lipopolysaccharide was considerably smaller and not used routinely. With 2 × 10⁵ lymphocytes/well, the background count of unstimulated lymphocytes in autologous serum in healthy dogs was usually between 100 and 400 counts/min (CPM), in clinically healthy cattle and horses from 200 to over 2000 CPM. Higher CPM were rarely detected without clinical disease. Increased background counts were often associated with viral infections, leukemias and lymphoreticular hyperplasias, decreased background counts were associated with various diseases. The stimulation indexes (SI) of healthy animals in autologous serum with Con A, (5 μg/ml) or PWM or PHA (1 μg/ml) were in the range from 100 to 1000 in the dogs, in the tens for Con A and in hundreds for PWM and PHA in horses and cattle. Increased SI were present during the incubation period of various diseases. Decreased SI were associated with numerous infectious and lymphoreticular diseases and were caused by any of the following: (1) the presence of serum immunosuppressive factor(s) in the patient serum, (2) the decreased response of lymphocytes to mitogens, or (3) increased mitogenicity of lymphocytes due to unidentified serum factors in absence of phytomitogens.     

紫锥菊多糖对鸡淋巴细胞体外增殖影响

为了评价紫锥菊多糖免疫增强效果,将4种浓度的紫锥菊多糖分别加入体外培养的鸡脾脏淋巴细胞中,培养44 h后,用MTT法测定淋巴细胞增殖变化。结果表明,4种浓度的紫锥菊多糖对鸡淋巴细胞均有促进增殖及协同的作用,其中在50-100μg/mL浓度范围内效果最为显著。     

Optimization of an antibiotic sensitivity assay for Mycoplasma hyosynoviae and susceptibility profiles of field isolates from 1997 to 2011

Mycoplasma hyosynoviae is a common agent responsible for polyarthritis leading to decreased production in swine herds worldwide. Antimicrobial agents are used to combat infections; however breakpoints for M. hyosynoviae have not yet been established. A number of methods have previously been utilized to analyze minimum inhibitory concentrations (MICs) for antibiotics against M. hyosynoviae; however these techniques as currently described are not easily standardized between laboratories. A dry microbroth dilution method was conducted to compare the minimum inhibitory concentrations (MICs) for 18 antibiotics, representative of different classes, against 24 recent isolates (23 field isolates and the type strain) of M. hyosynoviae. The MICs were determined using standard, commercially available 96-well Sensititre(?) plates containing various freeze-dried antibiotics at a range of concentrations appropriate to their potency. Clindamycin (CLI), a lincosamide antibiotic, showed the highest activity and most consistent inhibition for all isolates with an MIC(50) of ≤ 0.12 μg/ml. Tiamulin (TIA), a pleuromutilin derivative, exhibited an MIC(50) of ≤ 0.25 μg/ml. The isolates had similar levels of susceptibility to the quinolones, enrofloxacin (ENRO) and danofloxacin (DANO), exhibiting an MIC(50) of 0.25 μg/ml and 0.5 μg/ml, respectively. For the macrolides, the MIC(50) for tylosin (TYLT) and tilmicosin (TIL) was ≤ 0.25 μg/ml and ≤ 2 μg/ml respectively, but was ≤ 16 μg/ml for tulathromycin (TUL). For the aminoglycosides, the MIC(50) for gentamicin (GEN) was ≤ 0.5 μg/ml, while spectinomycin (SPE) and neomycin (NEO) had an MIC(50) of ≤ 4 μg/ml. The tetracyclines, oxytetracycline (OXY) and chlortetracycline (CTET) both had an MIC(50) of ≤ 2 μg/ml. Florfenicol (FFN) exhibited a MIC(50) of ≤ 1 μg/ml. All isolates were resistant to penicillin (PEN), ampicillin (AMP), ceftiofur (TIO), trimethoprim/sulfamethoxazole (SXT), and sulphadimethoxine (SDM) at all concentrations. Within the isolates tested, there was a range of sensitivity detected, with some isolates being overall more resistant while others appeared more susceptible. Further research is required to demonstrate how this MIC data correlates to clinical efficacy of the various antibiotics in the field.     

Serotypes, antimicrobial susceptibility, and minimal inhibitory concentrations of Actionbacillus pleuropneumoniae isolated from slaughter pigs in Taiwan (2002-2007)

In total, 211 isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during 2002-2007. Serotypes, antimicrobial susceptibility and minimum inhibitory concentration (MIC) values were determined for each isolate of A. pleuropneumoniae to 10 antimicrobial agents. Serovar 1 of A. pleuropneumoniae was predominant in Taiwan in 138 of the 211 isolates, followed by serovars 2 and 5. More than 90% of collected isolates were sensitive to ceftiofur, cephalothin, and chloramphenical. However, lincospectin and gentamicin were relatively less susceptible with sensitivities of only 2.4 and 5.7%, respectively. Additionally, ceftiofur had the highest in vitro activity with an MIC(50) of 2.2 μg/ml, followed by cephalothin (2.7 μg/ml) and chloramphenicol (7.9 μg/ml). Lincospectin had the least activity with MIC(50) and MIC(90) values of 73.9 and 114.5 μg/ml, respectively. The data indicate that ceftiofur and cephalothin were extremely active against A. pleuropneumoniae and with minimum MIC values. These drugs are suitable for controlling and treating hemorrhagic pleuropneumonia outbreaks in swine.