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Molecular biological studies on dolphin neutrophils

海豚中性粒细胞的分子生物学研究

基本信息

批准号：
13660327
负责人：
SAKAI Takeo
金额：
$ 2.3万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660327/
关键词：
Bottlenose dolphin Neutrophil Oxygen radicals NADPH oxidase Cytokines Molecular cloning Priming 腫瘍壊死因子α

项目摘要

The bottlenose dolphin NADPH oxidase components (gp91^<phox>, p22^<phox>, p40^<phox> and p67^<phox>) and cytokines (IL-1α, IL-1β, IL-1ra, IL-4, IL-8, IFNγ and TNFα) cDNA were cloned utilizing the RT-PCR with specific primers based on the conserved sequences of terrestrial mammalian homologs. The nucleic acid sequences of dolphin NADPH oxidase components and cytokines indicate high similarity (77.0-95.4%) with those of bovine homologs, and these deduced amino acid sequences were also similar to those of mammalian homologs.Dolphin neutrophils generated superoxide by the stimulation of phorbol ester and a specific inhibitor of NADPH oxidase suppresses its superoxide-forming activity. These results indicate that dolphin neutrophils possess NADPH oxidase activity to generate oxygen radicals. The respiratory burst (RB) accompanying oxygen radicals generation was induced by several stimulants, but not N-formyl-Met-Leu-Phe as well as bovine neutrophils. The RB of dolphin neutrophils stimulated with LPS and Staphylococcus aureus was inferior to those of bovine and human. This result suggests that the bactericidal activity of dolphin neutrophils was weaker than those of terrestrial mammals.The RB of dolphin neutrophils induced by various receptor-mediated agonists was enhanced by recombinant dolphin TNFα in a concentration-dependent manner. This TNFα-associated priming effect was hea-instable and not abolished by polymyxin B, indicating that the priming effect of recombinant dolphin TNFα preparations are not due to contamination with LPS derived from Escherichia coli. These data demonstrate that cytokines act as a modulator for innate immunity also in marine mammals.

宽吻海豚 NADPH 氧化酶成分（gp91^<phox>、p22^<phox>、p40^<phox> 和 p67^<phox>）和细胞因子（IL-1α、IL-1β、IL-1ra、IL-4、IL-8、IFNγ 和 TNFα）cDNA 采用基于 RT-PCR 的特异性引物进行克隆。陆生哺乳动物同源物的保守序列。海豚NADPH氧化酶成分和细胞因子的核酸序列与牛同源物具有高度相似性(77.0-95.4%)，并且这些推导的氨基酸序列也与哺乳动物同源物相似。海豚中性粒细胞通过佛波酯的刺激产生超氧化物，而NADPH氧化酶的特异性抑制剂抑制其产生。超氧化物形成活性。这些结果表明海豚中性粒细胞具有NADPH氧化酶活性以产生氧自由基。伴随氧自由基产生的呼吸爆发（RB）是由几种兴奋剂诱导的，但不是 N-甲酰基-Met-Leu-Phe 以及牛中性粒细胞。用LPS和金黄色葡萄球菌刺激的海豚中性粒细胞的RB低于牛和人的。这一结果表明，海豚中性粒细胞的杀菌活性弱于陆生哺乳动物。重组海豚TNFα以浓度依赖性方式增强各种受体介导的激动剂诱导的海豚中性粒细胞的RB。这种 TNFα 相关的启动效应是不稳定的，并且不会被多粘菌素 B 消除，这表明重组海豚 TNFα 制剂的启动效应不是由于来自大肠杆菌的 LPS 污染造成的。这些数据表明细胞因子在海洋哺乳动物中也充当先天免疫的调节剂。