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

脂肪酸环加氧酶（Fatty acid cycloxygenase， COX）启动生物活性前列腺素和凝血酶的合成，被认为是抗炎药物的靶点。这种酶在水疱腺、血小板、胃和其他各种动物组织中都有发现。1991年，该酶的同工酶（COX-2）被发现，并被发现是一种诱导酶。由于COX-2可被LPS和促炎细胞因子快速而短暂地诱导，因此在抗炎药物的开发中，COX-2比COX-1更受关注。本课题的目的是开发和建立实用方便的COX同工酶蛋白和mRNA的测定方法以及抗炎药物的体内作用。1)用具有特定序列的COX-2片段培养抗体。在先前针对牛水疱腺和人血小板酶的单克隆抗体中发现了COX-1选择性抗体。这些针对COX-1和COX-2的抗体可以应用于酶的Western blotting。2)尝试用RT-PCR扩增COX-1和COX-2 mrna，建立灵敏、便捷的检测方法。利用荧光标记制备非放射性探针。3)为了评估药物的体内作用，研究了疾病模型。采用角叉菜胶诱导大鼠胸膜炎进行COX-2研究。未处理大鼠（针对COX-1）和lps处理大鼠（针对COX-2）静脉注射花生四烯酸，定量测定给药后血液中花生四烯酸代谢物的变化。

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.