猪嗜中性粒细胞cAMP磷酸二酯酶的提取与活性检测  

Extraction and Activity Detection of cAMP-Phosphodiesterase from Porcine Neutrophils

作　　者：陈武[1] 姜代勋[1] 杨柳[2] 胡燕[1] 张春霞[1] 于同泉[2] 路苹[2] 

机构地区：[1]北京农学院动物科学技术系,北京102206 [2]北京市农业应用新技术重点实验室,北京102206

出　　处：《北京农学院学报》2003年第4期241-244,共4页Journal of Beijing University of Agriculture

基　　金：国家自然科学基金(30270978);北京市科技新星计划(9558100500);市教委;北京市农业应用新技术重点实验室资助

摘　　要：目的:提取动物体的cAMP磷酸二酯酶(PDE),并检测其活性,为兽医临床研究PDE活性的生理病理变化和中西药物对PDE活性调节作用建立实验方法。方法:猪血经过葡聚糖沉降,淋巴细胞分离液分离,特殊分离液洗涤和红细胞溶胀后,获得嗜中性白细胞。嗜中性白细胞破碎后释放PDE,通过反相高效液相色谱(RP HPLC)法测定反应体系中cAMP的含量,根据底物(cAMP)的减少量,确定嗜中性白细胞中PDE活性。结果:经显微镜计数嗜中性粒细胞纯度达95%;酶量在10～40μl间对cAMP的分解率为9 90%～31 39%,与PDE活性之间存在直线相关关系(r=0 99)。结论:猪嗜中性粒细胞中含有高纯度、高活性的cAMP PDE;高效液相色谱法可以准确测定PDE反应前后cAMP含量的变化;两种方法结合可以作为兽医临床研究机体PDE活性变化和中西药物对PDE活性调节作用的新方法。An experimental method was established by extracting cAMP-phosphodiesterase (PDE) from porcine neutrophils and detecting its activity, which can be used in veterinary clinic to explore its physiological and pathological changes, and to study the regulative effects of Chinese herbs and western medicine on its activity. Porcine neutrophils were obtained from porcine whole blood that was separated to remove erythrocytes with dextran and to separate lymphocytes with Ficoll Solution, and then cleaned with special cleaning fluid and distilled water to break the remnant erythrocytes. PDE was got from that the neutrophils were broken to release. The activity of PDE was decided upon the decreased content of cAMP, which was detected with reversed-phase high performance liquid chromatograph (HPLC). The results are that the purity of neutrophils reached 95% by counting them under microscope; and a well linear correlativity (r=0.99) existed between PDE activity and enzyme content ranged from 10μl to 40μl with 9.90%～31.39% of cAMP decomposition rate. The conclusions are that highly-pure and highly-active cAMP-PDE exists in porcine neutrophils; HPLC can be used to accurately detect the changes of cAMP content before and after PDE reaction.These two are combined as a new method to explore the changes of PDE activity in animal body and study the regulative effects of Chinese herbs and western medicine to PDE activity in veterinary clinic.

关 键 词：猪 嗜中性粒细胞 CAMP 磷酸二酯酶 提取技术 活性检测 

分 类 号：S852.33[农业科学—基础兽医学]