猪中性粒细胞磷酸二酯酶基因表达及活性研究.doc

猪中性粒细胞磷酸二酯酶基因表达及活性研究 姜代勋1,2，陈 武2，谷金妮1,2，许剑琴1，于同泉3，路 苹3，穆 祥4 （1中国农业大学动物医学院，北京 100094；2北京农学院动物科学技术系，北京 102206；3农业应用新技术北京市重点实验室，北京 102206； 4兽医学（中医药）北京市重点实验室，北京 102206） 摘要：【目的】通过对猪中性粒细胞磷酸二酯酶（PDE）基因表达、活性及特异性抑制剂实验检测，研究其存在的主要PDE亚型及在体外的活性，为探讨PDE在中性粒细胞的分布、活性变化及对cAMP、cGMP调节与中性粒细胞功能关系提供依据。【方法】采用反转录聚合酶链反应（RT-PCR）检测PDE基因在猪中性粒细胞的表达，以高效液相色谱（HPLC）检测环核苷酸在PDE反应前后的含量变化，计算PDE活性。【结果】在检测的18个PDE亚型中，PDE1B、2A、3A、3B、4A、4B、4C、4D、5A、7A、7B、8A、8B、9A、11A 共15个PDE mRNA在猪中性粒细胞表达，特异性抑制剂实验表明PDE4、PDE5分别为水解cAMP、cGMP的主要PDE亚型；cAMP/cGMP-PDE在10～40 μl范围内，与其活性存在良好的线性关系（r＝0.9929/0.9992）。【结论】猪中性粒细胞至少存在15个PDE亚型，其中PDE4、PDE5为主要存在的2个亚型，PDE样品量在一定范围内与其活性存在良好的线性关系，可作为筛选PDE型中性粒细胞功能调节剂的方法。 关键词：中性粒细胞；磷酸二酯酶；中国实验用小型猪；高效液相色谱 Study on Activity and Gene Expression of Phosphodiesterase in Porcine Neutrophils JIANG Dai-xun1,2, CHEN Wu2, GU Jin-ni1,2, XU Jian-qin1, YU Tong-quan3, LU Ping3, MU Xiang4 (1College of Veterinary Medicine, China Agricultural University, Beijing 100094; 2Department of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206; 3Key Laboratory of New Technology in Agriculture Application of Beijing Municipal, Beijing 102206; 4Beijing Key Laboratory of Veterinary Medicine (Traditional Chinese Medicine and Pharmacology), Beijing 102206) Abstract:【Objective】The objective was to study the main types and activity of phosphodiesterase (PDE) existed in porcine neutrophils through the examination of PDE gene expression, activity and selective inhibitor experiments. They investigated the relationship between PDE distribution, activity change, adjustment on cAMP and cGMP content and neutrophils function.【Method】PDE gene expression was detected by RT-PCR, and PDE activity was calculated by cyclic nucleotides content change examined with HPLC before and after the PDE reaction.【Result】mRNAs of PDE1B, 2A, 3A, 3B, 4A, 4B, 4C, 4D, 5A, 7A, 7B, 8A, 8B, 9A, 11A were expressed in porcine neutrophils, and selective inhibitor experiments indicated PDE4 and PDE5 were respectively the main