Present Concepts on the Inflammatory Modulators with Special Reference to Cytokines*

The pro- and anti-inflammatory cytokines create a network of interactions between cells that lead to both stimulatory and inhibitory responses that maintain an effective homeostatic regulation. The anti-inflammatory cytokines are a family of peptides that modulate the pro-inflammatory cytokine response. Cytokines act in concert with non-cytokine mediators, such as prostaglandin E₂, glucocorticosteroids, lipocortins, and catecholamines. This review highlights new developments in our understanding of the pathophysiology of inflammation and gives an example of a more recent approach to the modulation of acute systemic inflammatory disorders: activation of ₂-adrenergic receptors on macrophages. In this respect the potent ₂-adrenergic agonist clenbuterol seems of therapeutic interest.     

抗盐酸克伦特罗单链抗体基因的克隆、序列分析及表达

提取分泌抗盐酸克伦特罗单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,用一段柔性肽链-(G4S)3将VH和VL连接成ScFv。测序后经NCBI Blast分析,所得ScFv具有重组功能性鼠抗体可变区基因的特征。将所得目的基因与pET-22b( )连接,转化E.coli BL21,用IPTG诱导表达,表达蛋白经SDS-PAGE分析,37℃培养5 h表达量较大,25℃诱导表达以可溶性为主。     

低温与克伦特罗（CL）对肉仔鸡生产性能的影响

试验采用２×３（温度：１０ｖｓ２２℃，ＣＬ：３ｖｓ０１０＾－６）４重复完全交叉分组试验设计研究低温与克伦特罗对２９￣５６日龄ＡＡ雌性肉仔鸡生产性能的影响。结果表明；低温显著降低肉仔鸡生产性能，全期平均日采食量和耗料增重比分别增加５．９４％和９．０９％（Ｐ〈０．０１），平均日增重降低了２．９４％（Ｐ〈０．０５），且平均日增重还表现出对低温的适应。ＣＬ对试验全期肉仔鸡生产性能没有影响（Ｐ〉０．０５），     

金磁微粒介导的盐酸克伦特罗多克隆抗体的纯化

以重氮化法制备盐酸克伦特罗-牛血清白蛋白(CBL-BSA)抗原并免疫家兔制备多克隆抗体,将表面固定有BSA的金磁微粒(金磁微粒-BSA)与多克隆抗体反应,抗BSA抗体通过抗原抗体反应被吸附在金磁微粒表面,磁性分离,利用ELISA法对纯化前后抗CBL及抗BSA抗体的效价进行测定,确定最佳纯化条件,得到高纯度的抗CBL抗体。结果表明,通过一步反应．可实现载体蛋白BSA在金磁微粒表面的固定化,每毫克金磁微粒固定 BSA的容量可达236 μg;在最佳纯化条件(温度4C,反应时间2h,金磁微粒表面BSA质量与抗体质量比为7:1) 下,金磁微粒-BSA可有效地去除偶联物多克隆抗体中的抗BSA抗体,而纯化前后抗CBL抗体的效价基本保持不变,分离纯化效果较好。该方法可方便、快速地去除多克隆抗体中的无关抗体,对小分子半抗原多克隆抗体的纯化具有重要意义。     

克伦特罗残留检测试剂盒条件的优化及其应用

以CL-OVA为包被抗原,抗克伦特罗单克隆抗体为一抗,组建检测克伦特罗的竞争EL ISA试剂盒。以方阵滴定确定包被抗原最佳浓度(0.72μg.孔-1),抗克伦特罗单克隆抗体(2F12)最佳稀释度(1∶2 000),并建立EL ISA标准曲线,确定最小检测量为0.25 ng.mL-1。用该方法检测动物模型仔猪尿样中的盐酸克伦特罗,结果表明:停药后第1~7天尿样均为阳性,2周后有少量猪仍为阳性,而阴性对照猪尿样均为阴性。这表明建立的试剂盒对克伦特罗残留的检测具有广阔的应用前景。     

免疫金试纸法快速检测盐酸克伦特罗

试验选取自身蛋白制备克盐酸伦特罗克伦特罗载体抗原,免疫动物后产生高效价的特异性抗体,纯化抗体与胶体金结合成免疫金,制备成简易的检测试纸条。通过检测试纸条敏感性、保存期、金标抗体工作浓度选择、操作方法对比、实验检测及与单抗金标试纸条平行检测等试验,证明建立的免疫金试纸法快速检测盐酸克伦特罗具有实用性、可靠性和稳定性。自身全血清作载体可使抗盐酸克伦特罗的ELISA效价高达4万以上。免疫金试纸法对盐酸克伦特罗的最小检测量为40ng·mL-1,试纸条在室温保存150d不影响检测结果。实验检测86份ELISA试剂盒阴性样品和42份阳性样品,其符合率分别为100%和95.2%。市售单抗金标试纸条检测结果与ELISA法的阳性符合率为95%(19/20)。     

Clenbuterol Affects the Expression of Messenger RNA for Interleukin 10 in Peripheral Leukocytes from Horses Challenged Intrabronchially with Lipopolysaccharides

On four occasions, four horses with heaves and four horses with small airway inflammatory diseases inhaled 0.9% saline based aerosol mixtures with or without lipopolysaccharides (LPS). Prior to the first saline and LPS inhalation, horses were untreated, while three and a half days prior to the third and forth inhalation horses had received 0.8 μ g/kg clenbuterol intravenously twice daily. The messenger RNA (mRNA) expression of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and interferon- γ (IFN- γ) was investigated by RT-PCR, all of which were expressed in the white blood cells of samples collected. Inhalation of LPS only changed the cytokine expression profile of IL-10, IL-4 and TNF-α mRNA which were higher after challenge with LPS. However in those horses that were treated with clenbuterol the LPS-induced IL-10 mRNA expression was shown to be suppressed. Further changes in IL-4 and TNF-α were not significant. Thus the results of this study indicated that clenbuterol can modulate the expression of IL-10 mRNA in peripheral white blood cells in those horses with small airway diseases that have been exposed to LPS. van den Hoven, R., Duvigneau, J.C., Hartl, R.T. and Gemeiner, M., 2006. Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides. Veterinary Research Communications, 30(8), 921–928     

分子印迹固相萃取和丝网印刷电极联用技术检测猪尿中盐酸克伦特罗

实验采用本体热聚合的方式合成盐酸克伦特罗的分子印迹物(molecularly imprintedpolymers,MIPs),通过扫描电镜、吸附实验和分子印迹固相萃取研究该印迹材料的性能。用盐酸克伦特罗分子印迹材料作为固相萃取剂,优化固相萃取条件,采用差分脉冲伏安法(DPV)对洗脱液进行电化学性能测试。结果表明,此印迹物的平衡吸附常数(b)为5.14mL/mol,电化学传感器的电流值与盐酸克伦特罗的浓度在0.056～2.78mg/L浓度范围内呈现良好的线性关系,检测下限为0.005mg/L,盐酸克伦特罗在猪尿样品中的回收率为89.3%～99.6%。此萃取方法也呈现出良好的选择性和稳定性。     

Clenbuterol诱导载酯蛋白D在猪脂肪组织中表达加强

Clenbuterol(CLB,盐酸克伦特罗),俗称瘦肉精。由于其在动物体内广泛残留,可能导致食用此类产品的人中毒而被禁止使用,但已有研究表明该制剂可显著减少猪脂肪沉积,因此,对其分子机制进行深入研究将具有重要的理论与实践价值。本研究通过对家猪饲喂高剂量clenbuterol(≥1.5mg/kg体重),提高瘦肉率10%左右。通过实时荧光定量PCR技术对脂肪组织中与脂类代谢密切相关的4个基因研究发现,饲喂clenbuterol的猪脂肪组织内载酯蛋白D(apolipoprotein D,apoD)mRNA丰度增加超过2倍,而脂蛋白酯酶(lipoprotein lipase,lpl)、硬酯酰辅酶A去饱和酶(stearoyl-CoA desaturase,scd)和激素敏感脂酶(hormone sensitive lipase,hsl)mRNA丰度变化不明显。因此,推断clenbuterol通过改变部分脂代谢关键基因的表达来实现减少脂肪积累。载酯蛋白D可能是CLB应答基因之一。     

用HPLC-MS/MS研究动物毛发中克伦特罗的残留及代谢规律

研究建立了高效液相色谱-串联质谱(HPLC-MS/MS)测定毛发中盐酸克伦特罗残留的方法。猪毛发以0.1moL/L盐酸溶液提取,MCX固相萃取柱净化,流动相溶解定容。以SB-C18柱为分离柱,1%乙酸水溶液和甲醇(60∶40,V∶V)作为流动相,MS/MS进行定性和定量分析。在优化条件下的最低检出限为0.05μg/kg,定量限为0.2μg/kg。毛发样品中添加不同浓度盐酸克伦特罗,测得回收率在79.0%～86.2%之间,变异系数均低于15.2%。应用该方法研究了盐酸克伦特罗在猪毛发中的代谢并初步探讨其代谢机理。结果表明:在较高饲喂浓度(10mg/kg)时,用药5d后,黑色猪毛发有少量盐酸克伦特罗残留,而白色猪毛发中未检出。从第7天起猪毛发中盐酸克伦特罗蓄积量逐渐增加,最高蓄积浓度为4852.5μg/kg。