Development of cyclooxygenase-2 assay kits for new anti-inflammatory drugs

脂肪酸环加氧酶(COX)是生物活性前列腺素和血栓素类物质合成的起始酶，被认为是抗炎药物的靶点。在泡状腺、血小板、胃和其他各种动物组织中都发现了这种酶。1991年发现了该酶的同工酶(COX-2)，并发现它是一种诱导酶。由于COX-2是由内毒素和致炎细胞因子快速、短暂地诱导产生的，因此在抗炎药物的开发中，COX-2而不是COX-1受到了越来越多的关注。本课题的目的是建立实用简便的环氧合酶同工酶蛋白和信使核糖核酸测定方法以及抗炎药物体内效应的检测方法。1)用特定序列的环氧合酶-2片段制备抗体。COX-1选择性抗体是在以前提出的针对牛水泡腺和人血小板酶的单抗中发现的。这些COX-1和COX-2抗体可应用于酶的Western blotting。2)尝试通过RT-PCR扩增COX-1和COX-2的mRNAs，以建立敏感和方便的检测方法。正在利用荧光标记制备非放射性探针。3)为了评估药物的体内效应，对疾病模型进行了研究。角叉菜胶致大鼠胸膜炎用于COX-2研究。未治疗组(COX-1)和内毒素治疗组(COX-2)大鼠静脉注射花生四烯酸，定量测定给药后血中花生四烯酸代谢产物的变化。

Fatty acid cyclooxygenase (COX) initiates the synthesis of bioactive prostaglandins and thromboxanes, and has been considered as a target of anti-inflammatory drugs. The enzyme had been found in vesicular gland, platelets, stomach and various other animal tissues. In 1991 an isozyme of the enzyme (COX-2) was discovered, and found to be an inducible enzyme. Since COX-2 is induced rapidly and transiently by LPS and proinflammatory cytokines, more attentions have been directed to COX-2 rather than COX-1 in the development of anti-inflammatory drugs. The purpase of this project is to develop and establish practicallly convenient assay methods of the protein and mRNA of COX isozymes and the in vivo effect of anti-inflammatory drugs.1) Antibodies were raised with a COX-2 fragment with a specific sequence. COX-1 selective antibodies were found among monoclonal antibodies previously raised against the enzymes of bovine vesicular gland and human platelets. These antibodies for COX-1 and COX-2 could be applied to Western blottings of the enzymes.2) Amplification of mRNAs for COX-1 and COX-2 by RT-PCR was attempted to develop sensitive and convenient assays. Non-radioactive probes by the use of a fluorescent label are being prepared.3) For the assessment of in vivo effects of drugs, diseases models were investigated. Rat pleurisy induced by carrageenin was used for COX-2 studies. Non-treated (for COX-1) and LPS-treated (for COX-2) rats received arachidonic acid intravenously, and the changes of arachidonate metabolites in the blood were determined quantitatively upon administration of drugs.