Molecular biological studies on cyclooxygenases and lipoxygenases dynamics

本项目是对负责合成具有生物活性的花生四烯酸代谢物的环氧合酶和脂氧合酶进行分子生物学研究。(1)环氧合酶：小鼠成骨细胞系MC 3 T3-E1产生前列腺素E2，其环氧合酶被证明是由各种激素、生长因子和洋地黄素诱导的。进一步的研究表明，两个环氧合酶同工酶参与前列腺素E2的生产。环氧合酶-1仅被逐渐诱导2-3倍，而环氧合酶-2在1-2小时内迅速出现并增加15-60倍。(2)脂氧合酶：根据底物特异性和免疫原性，对白细胞型和血小板型12-脂氧合酶进行了比较。克隆了它们的cDNA和基因组DNA，并对其结构进行了鉴定。白细胞型酶不仅存在于白细胞中，而且存在于猪垂体、牛气管、犬脑和大鼠松果体中。血小板型12-脂氧合酶以前只在血小板中发现，最近在人表皮细胞中发现。建立了12-脂氧合酶mRNA的PCR扩增方法，并在3例骨髓增生性疾病患者的血小板中证实了低水平的12-脂氧合酶以及降低的酶活性和蛋白质。

This project is a molecular biological approach to cyclooxygenases and lipoxygenases which are responsible for the syntheses of bioactive arachidonate metabolites.(1) Cyclooxygenases : A murine osteoblastic cell line MC3T3-E1 produces prostaglandin E2, and its cyclooxygenase enzyme was shown to be induced by various hormones, growth factors and prostaglandins. Further studies demonstrated the involvement of two cyclooxygenase isozymes in the prostaglandin E2 production. Cyclooxygenase-1 was induced gradually only by 2-3 fold, whereas cyclooxygenase-2 appeared rapidly within 1-2 h and increased by 15-60 fold.(2) Lipoxygenases : The leukocyte-type and platelet-type of 12-lipoxygenase were distinguishied in terms of substrate specificity and immunogenicity. Their cDNAs and genomic DNAs were cloned, and their structures were elucidated. The leukocyte-type enzyme was found not only in leukocytes but also in porcine pituitary, bovine trachea, canine brain and rat pineal gland. The platelet-type 12-lipoxygenase, which had been found only in platelets, was recently found in human epidermal cells. A method for PCR-amplification of 12-lipoxygenase mRNA was established, and a low level of 12-lipoxygenase together with a decreased enzyme activity and protein was demonstrated in the platelets of three patients with myeloproliferative diseases.