Spin-trapping detection of nitric oxide (NO) production from macrophage during matuation

自旋捕获检测巨噬细胞成熟过程中一氧化氮 (NO) 的产生

• 批准号: 07660387
• 负责人: KUWABARA Mikinori
• 金额: $ 1.66万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660387/
• 关键词: Nitric oxide; Macrophage; MGD; ESR; Interferon-gamma; Lipopolysaccharide; lactoferrin; spin-trapping; 単球; 一酸化窒素; スーパーオキシド; スピントラッピング; 電子スピン共鳴法; メチルグルカミンジカルバメート

In order to investigate nitric oxide (NO) production from macrophage during maturation, peneatrial macrophage was isolated from thioglycolate-stimulated mouse and the production of NO after stimulation by interferon-gamma (INF-gamma), lipopolysaccharide (LPS) and lactoferrin (LF) was measured by the method combining electron-spin-resonance spectroscopy (ESR) and spin-trapping. The complex (MGD-Fe) of Fe^<2+> and N- (Dithiocarbonyl) -N-methylglucamine was used as spin-trapping reagent. Triplet ESR signal due to MGD-Fe-NO,which was produced by the reaction of NO and MGD-Fe, was time-dependently observed in LPS+Inf-gamma-stimulated macrophage. The NO production was also confirmed the increased-responses of NO_2^-+NO_3^-, metabolites of NO,by colorimetric method using Griess reagents. It was also shown that NO synthase detected by Western blotting was induced by these stimulation. Furthermore, LF promoted the NO production and iNOS induction in INF-gamma-stimulated macrophage, whereas LF itself did not produce any NO production in macrophage. These results was suggested that the maturation of macrophage was induced by LF which was produced from neutrophil in inflammation site as well as various cytokines which was produced from various lymphocytes.

为了研究巨噬细胞成熟过程中一氧化氮（NO）的产生，分离了巯基乙酸刺激的小鼠心房巨噬细胞，用电子自旋共振波谱（ESR）和自旋捕集技术测定了干扰素-γ（INF-γ）、脂多糖（LPS）和乳铁蛋白（LF）刺激后巨噬细胞NO的产生。Fe^<2+>与N-（二硫代羰基）-N-甲基葡萄糖胺的络合物（MGD-Fe）被用作自旋捕获试剂。在LPS+ INF-γ刺激的巨噬细胞中，观察到由NO与MGD-Fe反应产生的MGD-Fe-NO的三重ESR信号，其具有时间依赖性。用Griess试剂比色法测定NO代谢产物NO_2^-+NO_3^-的浓度，证实了NO的产生。Western印迹法检测到NO合酶也被这些刺激诱导。此外，LF促进了INF-γ刺激的巨噬细胞的NO产生和iNOS诱导，而LF本身不产生任何NO。提示炎症部位中性粒细胞产生的LF和淋巴细胞产生的多种细胞因子共同诱导巨噬细胞成熟。

项目成果

Ohshima, H.: "Damage induced by hydroxyl radicals generated in the hydration layer of gamma-irradiated frozen aqueous solution of DNA." Jouranal of Radiation Research. 37. 199-207 (1996)

Ohshima, H.：“伽马射线照射的冷冻 DNA 水溶液的水合层中产生的羟基自由基引起的损伤。”

Kuwabara, M.: "Lipid peroxide levels and superoxide-scavenging abilities of sera obtained from hotbred (Thoroughbred) horses." Journal of Veterinary Medical Science. 58. 97-101 (1996)

Kuwabara, M.：“从纯种马获得的血清的脂质过氧化物水平和超氧化物清除能力。”

Nagahata, H., Higutchi, H., Goji, N., Noda, H.and Kuwabara, M: "Functional characteristics of enhanced Fc receptor expression of beta2 integrin-deficient bovine mononuclear phagocyte." Microbiol.Immunol.40. 389-395 (1996)

Nagahata, H.、Higutchi, H.、Goji, N.、Noda, H. 和 Kuwabara, M：“β2 整合素缺陷型牛单核吞噬细胞 Fc 受体表达增强的功能特征。”

Nagahata, H.: "Functional characteristics of enhanced Fc receptor expression of β2 integrin-deficient bovine mononuclear phagocyte." Microbiology and Immunology. 40. 389-395 (1996)

Nagahata, H.：“β2 整合素缺陷的牛单核吞噬细胞 Fc 受体表达增强的功能特征。微生物学和免疫学。” 40. 389-395 (1996)

Kuwabara, M., Inukai, N., Inanami, O., Miyake, Y., Tsunoda, N., Maki, Y.and Sato, F: "Lipid peroxide levels and superoxide-scavenging abilities of sera obtained from hotbred (Thoroughbred) horses." J.Vet.Med.Sci.58. 97-101 (1996)

Kuwabara, M.、Inukai, N.、Inanami, O.、Miyake, Y.、Tsunoda, N.、Maki, Y.和 Sato, F：“从杂交种（纯种马）获得的血清的脂质过氧化物水平和超氧化物清除能力