[目的]研究猪繁殖与呼吸综合征病毒(PRRSV)单独和联合脂多糖(LPS)刺激对肺脏募集中性粒细胞(Neu)的影响,以及猪肺微血管内皮细胞(MVECs)在其中的作用.[方法]取约10日龄仔猪的肺脏组织,采用Ⅱ型胶原酶消化和差速贴壁法,分离培养原代MVECs,采用密度梯度离心法分离猪外周血Neu;以PRRSV HN株或PRRSV-LPS刺激Neu和M VECs后,将Neu加入培养M VECs的培养板,孵育1h,4％多聚甲醛固定,瑞氏染色,观察并计数分析各组黏附的Neu数量;采用免疫细胞化学染色法检测MVECs对P-选择素和E-选择素的表达;采用虎红溶液染色法测定分析P-选择素和E-选择素抗体封闭MVECs时,PRRSV HN株或PRRSV-LPS刺激对Neu黏附于MVECs数量的影响.[结果]分离培养的MVECs呈CD34免疫荧光染色阳性,阳性率约为92％;PRRSV HN株刺激18h,黏附于MVECs的Neu数量显著增加(P＜0.05);PRRSV-LPS联合刺激时,黏附的Neu数量显著多于LPS单独刺激(P＜0.05);PRRSV或PRRSV-LPS刺激Neu和MVECs二者时,黏附Neu数量的增加比单独刺激M VECs或Neu更明显;免疫细胞化学染色显示,MVECs强阳性表达P-选择素和E-选择素;用P-选择素和E-选择素抗体封闭MVECs时,PRRSV或PRRSV-LPS刺激诱导的Neu黏附增加呈不同程度下降,E-选择素封闭时具有显著差异(P＜0.05).[结论]PRRSV感染能促进Neu与MVECs黏附,亦能增加后继LPS刺激时黏附的Neu数量,MVECs表达的E-选择素是介导PRRSV致Neu黏附增加的重要分子之一.

旨在通过蛋白组学探索益母草水煎液对人真皮微血管内皮细胞(human dermal microvascular endothelial cells,HDMECs)凝血与抗凝血相关因子表达的调节作用.MTT法筛选益母草水煎液对HDMECs的安全浓度;使用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)技术分析 50 和100μg·mL-1益母草水煎液作用24 h后HDMECs蛋白表达谱的变化,通过对比各组蛋白谱的变化筛选出差异显著的相关通路,分析该通路中筛选到的可信差异表达蛋白(differentially expressed proteins,DEPs),并选取可信DEPs中与凝血与抗凝血相关的拮抗因子进行RT-PCR和ELISA验证.结果表明:1 mg·mL-1以下益母草水煎液对HDMECs无毒副作用;益母草水煎液能够同时调节与凝血相关的血小板活化等过程和与抗凝血相关的肝素结合过程.对筛选出的补体和凝血级联通路中的5种可信DEPs分析显示,与空白组相比,50μg·mL-1益母草水煎液组凝血酶原(F2)、抗凝血酶-Ⅲ(AT-Ⅲ)、组织型纤溶酶原激活剂(t-PA)、凝血因子V(F5)和激肽原(KNG)同时显著下调,100μg·mL-1益母草组F2、t-PA、AT-Ⅲ、KNG显著下调;与50 μg·mL-1益母草水煎液组相比,100μg·mL-1益母草水煎液组F2、AT-Ⅲ、t-PA、F5和KNG等凝血级联相关因子均显著升高.结果提示,益母草水煎液可显著改变HDMECs蛋白表达谱,并可能通过调控补体和凝血级联通路相关因子F2、AT-Ⅲ、t-PA、F5、KNG蛋白的表达对凝血与抗凝血相关拮抗因子发挥双向调控作用.

中(兽)医药学是我国的传统医学宝库之一.2020年6月总书记强调,要加强古典医籍精华的梳理和挖掘,建设一批科研支撑平台,改革完善中药审评审批机制,促进中药新药研发和产业发展,要强化中医药特色人才建设,打造一支高水平的国家中医疫病防治队伍.兽医学(中医药)北京市重点实验室始建于2006年12月,经北京市科学技术委员会和北京市教育委员会批准成立,依托单位为北京农学院,是国内较早从事中兽医药学研究的省部级重点实验室.实验室的主要研究方向包括:(1)防控畜禽细菌性疾病中药机制及药物研发;(2)防控畜禽病毒病的中药机制及药物研发;(3)宠物疾病与中兽医防控;(4)经络本质的探索研究.

1背景 1.1我国以疫苗免疫为主体的防疫政策需要重新评估 现状:老病不断、新病频发、不能防治无疫苗的疫病,这些绑架了养殖企业,给养殖企业造成很多问题. 原因:有利益因素;有政策因素:病原管控不够、疫苗安全性评价和监控不严、忽视了治疗性药物的研发.另外还有技术因素:包括疫苗问题、普免的问题等等.

目的 明确两种微血管内皮细胞(MVECs)糖链表达的特点.方法 复苏冻存的大鼠空肠黏膜MVECs;取3日龄SPF仔猪肺组织,采用胶原酶消化法和差速贴壁法分离培养猪肺MVECs;采用凝集素细胞化学方法,检测刀豆凝集素(Con A)、菜豆红细胞凝集素(PHA-E)、蓖麻凝集素Ⅰ(RCA-Ⅰ)、番茄凝集素(LEL)、黑接骨木凝集素(SNA)、荆豆凝集素Ⅰ(UEA-Ⅰ)、麦胚凝集素(WGA)和双花扁豆凝集素(DBA)共8种凝集素在两种MVECs的染色情况.结果 大鼠空肠黏膜MVECs对Con A、WGA和LEL呈强阳性着色,PHA-E、SNA和RCA-Ⅰ呈中等阳性着色,DBA呈弱阳性着色,UEA-Ⅰ呈阴性着色;猪肺MVECs对Con A和PHA-E呈强阳性着色,RCA-Ⅰ呈中等阳性着色,LEL和SNA呈弱阳性着色,UEA-Ⅰ、WGA和DBA呈阴性着色.结论 两种MVECs的糖链表达具有显著异质性,大鼠空肠黏膜MVECs和猪肺MVECs均不同水平地表达甘露糖、半乳糖、1,3-N-乙酰葡糖胺和唾液酸,N-乙酰葡糖胺和N-乙酰半乳糖胺表达于前者不表达于后者,岩藻糖在两者均不表达.

Intestinal microvascular endothelial cell (IMVEC) is a fundamental and essential component of gut-vascular barrier which is closely associated with intestinal disorders However, there is still a lack of established intestinal microvascular endothelial cell line. In the present study, a newly established rat intestinal microvascular endothelial cell line termed RIMVEC-11 was described and characterized which has been stably cultured for more than 90 passages so far. RIMVEC-11 was characterized by endothelial features with the cobblestone morphology under light microscopy, the Weibel-Palade body and rich vesicles in the cytoplasm on the ultrastructural level, and positive endothelial specific markers CD31 and von Willebrand factor by immunocytochemistry analysis. Meanwhile, RIMVEC-11 maintained the fundamental physiological function of the microvascular endothelial cells. Tube formation assay confirmed that RIMVEC-11 retained the potential for capillaries formation. Scratch assay confirmed the endothelial cell migration potential of RIMVEC-11. Thus, a novel IMVEC cell line RIMVEC-11 was established, which could be used as a promising model for the gut-vascular barrier research.

Objectives : Microvascular endothelial cells (MECs) have been proved by increasing studies to play important roles in the process of endocrine, immune response, and pathogenic microorganism infection. However, most types of MECs have a limited number of divisions. Therefore, the immortalization of primary MECs may provide a better cell model for research. And the present research is aimed to establish an immortal human dermal microvascular endothelial cells (HDMECs). Methods : To immortalize HDMECs, the telomerase reverse transcriptase (hTERT) gene was transferred into the primary HDMECs by lentiviral infection. The passages of HDMECs transfected with hTERT or without hTERT were analyzed. At the same time, the relative telomerase activity and telomere length in HDMECs transfected with hTERT were detected by RT-PCR assay. And the β-galactosidase (β-GAL) activity in HDMECs transfected with hTERT was detected by ELISA kits. Finally, karyotype and tube formation analysis were used to evaluate the effects of transfection with hTERT on the characteristics of HDMECs. Results: The results showed that the number of passages of HDMECs transfected with hTERT was significantly increased. The telomerase activity of HDMECs transfected with hTERT gene was enhanced, and β-GAL activity was significantly reduced. Moreover, the transfection of hTERT gene has almost no effect on the karyotype and tube formation of HDMECs. Conclusion: These data indicate that transfection of hTERT gene could successfully enhance the cleavage ability of HDMECs, and the characteristics of hTERT-HDMECs remain almost unchanged.

为了评估大肠杆菌O139及其所产志贺样毒素二型变异体(SLT-IIe)对猪临床相关指征的影响是否一致,试验选取23日龄仔猪15头,随机分为对照组、细菌组、毒素组,每组5只.通过观察和触摸的方法采集各组仔猪的临床症状,通过剖检及HE染色观察各组仔猪脏器的病理变化.结果 表明:与对照组相比,毒素组和细菌组呈现面部水肿、共济失调等仔猪水肿病特征性症状,肠腔积液,结肠系膜呈透明胶冻样水肿,胃壁水肿;组织学检查发现模型组小肠绒毛变短,肠黏膜下层及固有层水肿.研究表明,用大肠杆菌O139和SLT-IIe均成功诱导了仔猪水肿病,且用细菌和SLT-IIe诱导的发病猪各项临床指征基本一致.

1养殖业为何需要中兽药 1.1是从源头上保障畜禽产品安全的需要 1.1.1西兽药不能保障畜禽产品安全 西医药学的基本理论:细胞是构成动物体的结构和功能单位,致病因子导致细胞的结构和功能异常是疾病发生的根本原因,因此,消除致病因子和调控疾病细胞的功能使之恢复正常是治疗疾病的基本措施.

本试验旨在探讨温肾益精散对断奶仔猪生长性能和免疫活性的影响.本试验选取35日龄体重相近、健康的断奶仔猪120头,随机分为3组(每组4个重复,每个重复10头猪).对照组饲喂基础饲粮,高剂量组、低剂量组分别饲喂添加0.5％、0.25％温肾益精散的试验饲粮,试验期40d.结果 表明:温肾益精散高、低剂量组的平均日增重显著高于空白对照组(P＜0.05),高剂量组最高;高、低剂量组的胰岛素样生长因子1 (IGF-1)和生长激素(GH)含量显著高于空白对照组(P＜0.05),而生长抑素(SST)含量显著低于空白对照组(P＜0.05);高、低剂量组的IgA、IgG、IgM、IL-2、IL-4含量显著高于空白对照组(P＜0.05),高剂量组最高.由此可见,饲粮添加适量温肾益精散可提高断奶仔猪的生长性能和免疫功能,在本试验条件下断奶仔猪饲粮中添加0.5％的温肾益精散效果为最佳.

Gut microbiota serves as a critical indicator for gut health during treatment of pathogenic bacterial infection. Both Pulsatilla Decoction (abbreviated to PD, a traditional Chinese medicine compound) and Levofloxacin Hydrochloride (LVX) were known to have therapeutic effects to intestinal infectious disease. However, the changes of gut microbiota after PD or LVX treatment remain unclear. Herein, this work aimed to investigate the changes of intestinal flora after PD or LVX therapy of Escherichia coli infection in rats. Results revealed that PD exhibited a valid therapeutic approach for E. coli infection via the intestinal protection, as well as the inhibited release of IL-8 and ICAM-1. Besides, PD was beneficial to rebuild the gut microbiota via restoring Bacteroidetes spp in the composition of the gut microbiota. Comparatively, LVX treatment promoted the infection and ravaged gut microbiota by significantly decreasing Bacteroidetes and increasing Firmicutes. These findings not only highlight the mechanism of Chinese herbal formula, but extend the application of PD as veterinary medicine, feed additive and pre-mixing agent for improving the production of animal derived foods.

BACKGROUND/PURPOSE:Synchronous microvascular vasomotion was detected at acupoints in our previous human study. This present study aimed to characterize the skin microvascular vasomotion at acupoints on the twelve meridians of beagle dogs.MATERIALS AND METHODS:Two acupoints were selected on each meridian and exactly located at the rosy red spots by an electrochemical color-appearing method, where the electrical resistance was measured. The skin blood flow at acupoints was recorded by laser Doppler flowmetry (LDF), and microvascular vasomotion was analyzed according to LDF waveforms.RESULTS:The skin electrical resistance at acupoints was significantly lower than that at control non-acupoints. The LDF waveforms at acupoints were sinusoidal, which showed the synchronization of the microvascular vasomotion. The spectral analysis revealed that the vasomotion frequencies at acupoints on the same meridian were identical but not among different meridians, and the frequencies on the twelve main meridians displayed a constant order.CONCLUSION:The skin microvascular vasomotion is synchronous at acupoints of beagle dogs and has a specific frequency along the meridian, and the electrochemical color-appearing method is a feasible strategy for the precise and visual location of acupoints. The study provides evidence for the universality of synchronous vasomotion of skin microvessels at acupoints.

Abstract Background The PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs. Methods The susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody. Results Surface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells. Conclusions Taken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

本试验旨在探究大肠杆菌志贺样毒素Ⅱ型变异体(SLT-Ⅱe)处理后,猪小肠微血管内皮细胞(PIMVECs)分泌的肿瘤坏死因子α(TNF-a)、P选择素(P-selectin)、一氧化氮(NO)、细胞间黏附分子1(ICAM-1)和内皮素(ET)水平的变化.采用超速离心法提取SLT-Ⅱe,采用BCA试剂盒测定其质量浓度,使用SDS-PAGE及Westernblotting验证蛋白纯度;用WST-1法从0.1、0.5、1、5、10 μg/mL SLT-Ⅱe和0、2、4、8、12、24 h中筛选出SLT-Ⅱe对PIMVECs最佳作用浓度与时间;ELISA法检测对照组(0 μg/mL SLT-Ⅱe)和试验组(1 μg/mL SLT-Ⅱe)细胞上清中TNF-a、P-selectin、NO、ICAM-1和ET含量的变化.结果 表明:所提蛋白为SLT-Ⅱe,蛋白含量为721.75 μg/mL;1 μg/mL的SLT-Ⅱe作用8h后可极显著降低PIMVECs的活性(P＜0.01);1μg/mL的SLT-Ⅱe作用9和12h时PIMVECs的TNF-a分泌量显著升高(P＜0.05),NO/ET的值显著高于对照组(P＜0.05),作用6、9、12 h时P-selectin的含量显著升高(P＜0.05),作用12 h时的ICAM-1分泌量显著升高(P＜0.05).综上所述,SLT-Ⅱe浓度为1 μg/mL时可以诱导PIMVECs分泌的TNF-a、P-selectin、ICAM-1以及NO/ET上升,为研究仔猪水肿病发病机制提供参考.

为了弥补口蹄疫疫苗的免疫缺陷,为寻找新型低毒的口蹄疫疫苗中药佐剂提供试验依据,试验将体外培养的大鼠心肌膜微血管内皮细胞(MVECs)分为空白对照组(加入正常培养液)、口蹄疫疫苗组(加入混有口蹄疫疫苗稀释液的培养液)、口蹄疫疫苗+川芎嗪组(加入混有口蹄疫疫苗稀释液与川芎嗪溶液的培养液),分别测定口蹄疫疫苗与口蹄疫疫苗+川芎嗪对心肌膜MVECs分泌一氧化氮(N0)和血管内皮素1(ET-1)的影响.结果 表明:口蹄疫疫苗组心肌膜MVECs分泌NO和ET-1的量较空白对照组极显著升高(P＜0.01),但不是同比例升高,导致ET-1/NO的值偏离空白对照组接近2倍;而口蹄疫疫苗+川芎嗪组心肌膜MVECs分泌ET-1和NO的量较口蹄疫疫苗组显著或极显著降低(P＜0.05或P＜0.01),较空白对照组仍明显升高,但ET-1/NO的值比口蹄疫疫苗组降低,较接近空白对照组.说明中药川芎嗪保护了被口蹄疫疫苗损伤的心肌膜MVECs.

Migration of neutrophils across endothelial barriers to capture and eliminate bacteria is served as the first line of innate immunity. Bacterial virulence factors damage endothelium to produce inflammatory cytokines interacts with neutrophils. However, the mechanisms that behind endothelial-neutrophil interaction impact on the bactericidal activity remain unclear. Therefore, we aimed to find the target proteins on endothelial cells that triggered the bactericidal activity of transendothelial neutrophils. Herein, we built the infected models on rats and endothelial-neutrophil co-cultural system (Transwell) and discovered that endothelial-derived IL-1α promoted the survival of rats under Escherichia coli infection and enhanced the bactericidal activity of transendothelial neutrophils in vivo and in vitro. Results further showed that IL-1α was inhibited by lipopolysaccharide (LPS) in the endothelial-neutrophil interaction. We found that LPS mainly damaged cell membrane and induced cell necrosis to interrupt neutrophil migration from endothelial barrier. Thus, we used the isobaric tags for relative and absolute quantification (iTRAQ) method to identify different proteins of endothelial cells. Results showed that IL-1α targeted cellular plasma membrane, endoplasmic reticulum and mitochondrial envelope and triggered eleven common proteins to persistently regulate. During the early phase, IL-1α triggered the upregulation of cell adhesion molecules (CAMs) to promote neutrophil adhesion, while oxidative phosphorylation was involved in long time regulation to induce transmigration of neutrophils against bacteria. Our results highlight the critical mechanism of endothelial-derived IL-1α on promoting bactericidal activity of transendothelial neutrophils and the findings of IL-1α triggered proteins provide the potentially important targets on the regulation of innate immunity.

[目的]为研究白薇水煎液对LPS诱导的鸡肠炎模型的治疗效果.[方法]将60只28日龄肉鸡分为生理盐水组、LPS组和LPS+白薇水煎液组.连续3d对各组灌服不同浓度的白薇水煎液(3、6、9 g/kg)及6mL/kg的生理盐水.3d后,除生理盐水组按1 mL/kg注射生理盐水外,其余各组腹腔注射2 mg/kg的LPS,12 h后收集鸡十二指肠进行组织病理切片,观察其肠道病理变化,利用ELISA的方法检测血清中淀粉样蛋白A(SAA)、卵转铁蛋白(OVT)、α-1-酸性糖蛋白(AGP)和白介素1β(IL-1β)表达水平.[结果]使用白薇水煎液(6 g/kg)后,可明显减轻LPS导致的肠绒毛黏膜上皮组织结构的损伤和炎性细胞浸润,并可显著或极显著抑制SAA (P＜0.01)、OVT (P＜0.05)、AGP (P＜0.01)、IL-1β (P＜0.01)炎性因子的表达.[结论]结果提示白薇水煎液(6 g/kg)具有较好的肠黏膜保护作用,抑制LPS刺激后血清中SAA、OVT、AGP、IL-1β的高表达.

Background/purpose: Skin microvessels at acupoints have been documented to be more abundant and well-organized, and the synchronous microvascular vasomotion was detected at acupoints in our previous human study. This present study aimed to characterize the skin microvascular vasomotion at acupoints on the twelve meridians of beagle dogs. Materials and Methods: Two acupoints were selected on each meridian, and exactly located at the rosy red spots by an electrochemical color-appearing method, where the electrical resistance was measured. The skin blood flow at acupoints was recorded by laser Doppler flowmetry (LDF), and microvascular vasomotion was analyzed according to LDF waveforms. Results: The skin electrical resistance at acupoints was significantly lower than that at control non-acupoints. The LDF waveforms at acupoints was sinusoidal, which showed the synchronization of the microvascular vasomotion. The spectral analysis revealed that the vasomotion frequencies at acupoints on the same meridian were identical but not among different meridians, and the frequencies on the twelve main meridians displayed a constant order. Conclusion: The skin microvascular vasomotion is synchronous at acupoints of beagle dogs and has a specific frequency along the meridian, and the electrochemical color-appearing method is a feasible strategy for the precise and visual location of acupoints. The study provides evidence for the universality of synchronous vasomotion of skin microvessels at acupoints and contributes to clarifying the essence of acupoints and their effect mechanism.

为探究自拟复方中草药的临床疗效,试验选用60只10-12 g雄性Bal/bc小鼠随机分成4组,空白对照组和低、中、高剂量组,分别为含10g/kg、20g/kg、40g/kg的粉碎复方中草药鼠粮,每组15只,试验周期15天.结果 表明:与对照组相比,中剂量组在极显著提高小鼠的日增重(P＜0.01)同时料肉比极显著下降(P＜0.01)并且生长激素释放肽释放量显著升高(P＜0.05),高剂量组平均日增重显著升高(P＜0.05);血液生化指标中,低剂量组与对照组相比尿酸和甘油三酯含量显著降低(P＜0.05).与对照组相比,各组小鼠脾淋巴细胞在刀豆蛋白处理24 h后,低剂量组和中剂量组增殖率显著提高(P＜0.05),48 h后中剂量组淋巴细胞增殖率显著提高(P＜0.05).综上,说明中剂量组即含有20 g/kg复方中草药鼠粮组可以显著提高Bal/bc小鼠的生长性能,并且可以一定程度上提高其免疫功能.

胚胎干细胞(ESCs)作为一种全能性细胞,可体外培养无限增殖,并自我更新和向多种细胞分化.对于不能再生的心肌细胞,若能通过诱导ESCs分化为纯度高的心肌细胞,参与心肌的再生及修复,在加快其在临床上的应用及治疗心脏疾病方面具有极其重要意义.淫羊藿苷(ICA)作为一种生物活性较强的中药提取物,可改善心脑血管的血流量、改善心血管系统功能、诱导肿瘤细胞及干细胞分化等,还可诱导ESCs分化为心肌细胞.