猪中性粒细胞磷酸二酯酶基因表达及活性研究

 【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。

Study on Activity and Gene Expression of Phosphodiesterase in Porcine Neutrophils

Abstract:【OBJECTIVE】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 for the investigation of relation between PDE distribution, activity change, adjustment on cAMP and cGMP content and neutrophil function;【METHOD】PDE gene expression was detected by RT-PCR, and PDE activity was calculated by cylic nucleotides content change examined with HPLC before and after the PDE reaction;【RESULTS】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 types to hydrolyze cAMP and cGMP; cAMP/cGMP-PDE in the extent of 10 µL ~ 40 µL had a good linear correlation with its activity (r＝0.9929, 0.9992);【CONCLUSION】there are at least 15 PDE types existed in porcine neutrophils, in which PDE4 and PDE5 are the two main types; PDE volume has a good linear correlation with its activity, to some extent, which supply the base for the choose herbal regulators of neutrophil function of PDE type.