一种检测巨噬细胞内NADPH氧化酶活性的新方法

目的建立一种检测活细胞内ＮＡＤＰＨ氧化酶活性的新方法。方法用超氧离子物荧光探针ＨＥ标记巨噬细胞后,在激光扫描共聚焦显微镜下,用最适发射波长为５３０ｍｍ的检测器１检测巨噬细胞内ＨＥ的荧光强度变化。结果检测器１可检测单个和多个巨噬细胞内ＨＥ的荧光强度变化,ＰＭＡ刺激后巨噬细胞内ＨＦ荧光强度增强,ＮＡＤＰＨ氧化酶活性升高;地塞米松使巨噬细胞内ＨＥ荧光强度减弱,ＮＡＤＰＨ氧化酶活性降低：经地塞米松处理０．５ ｈ, ＰＭＡ刺激引起的巨噬细胞内 ＨＥ荧光强度增强幅度减小, ＮＡＤＰＨ氧化酶活性变化减小。结论激光扫描共聚焦显微术结合荧光标记技术可原位实时观察活细胞内ＮＡＤＰＨ氧化酶活性的变化。

A new way for detection of NADPH oxidase activity in the macrophages

Objective To establish a new method for detecting NADP oxidase activity in the macrophages. Methods The superoxide-sensitive fluorescence probe HE was used to mark the macrophages, and the alteration of the fluorescence density was observed with detector 1 (optimal emission wavelength 530 nm) of the laser scanning confocal microscope (ACAS570). Results Superoxide generated by NADPH oxidase can oxidize HE into fluorescent product ethidium bromide, and the alterations in the fluorescenee density of HE denoting the changes in NADPH oxidase activity of the macrophages can be monitored by detector 1 PMA increased the HE fluorescence of the macrophages, indicating that the NADPH oxidase activity was enhanced, dexamethasone reduced the HE fluorescence, indicating NADPH oxidase activity was decreased, Pretreated with dexamethasone for 0.5 h, the magnitude of the increase in HE fluorescence induced by PMA was lowered, indicating the degree of the enhancement of NADPH oxidase activity lowered also. Conclusion By using laser scanning confocal microscope and superoxide sensitive fluorescence probe HE, the kinetic variations in NADPH oxidase activity of the attached macrophages could be observed originally and continuously in single cell or multiple cells,