大鼠突触体中氧活性物质形成的灵敏快速定量分析
Sensitive and rapid quantitation of oxygen reactive species formation in rat synaptosomes.
作者信息
Lebel C P, Bondy S C
机构信息
Department of Community and Environmental Medicine, Southern Occupational Health Center, University of California, Irvine, CA 92717, U.S.A.
出版信息
Neurochem Int. 1990;17(3):435-40. doi: 10.1016/0197-0186(90)90025-o.
The formation of oxygen reactive species in response to oxidative stimuli was measured in rat synaptosomes. Studies employed the non-fluorescent probe 2?,7?-dichlorofluorescin diacetate (DCFH-DA), which after de-esterification is oxidized in the presence of oxygen reactive species to the highly fluorescent 2?,7?-dichlorofluorescein (DCF). Oxygen reactive species formation, as measured by DCF fluorescence, was stimulated by ascorbate and/or FeSO(4), and xanthine/xanthine oxidase under various buffering conditions. These agents all increased DCF formation in Tris, HEPES and phosphate buffer. Ascorbate also stimulated the formation of DCF in a concentration-dependent manner. The presence of Ca(2+) in HEPES buffer did not enhance or diminish the effects of ascorbate/FeSO(4) on DCF formation. Deferoxamine inhibited the ascorbate/FeSO(4)-induced stimulation of DCF formation, but xanthine/xanthine oxidase-induced stimulation was not affected by pretreatment with superoxide dismutase. Results indicate that DCF fluorescence is a sensitive, quantitative and direct measure of oxygen reactive species formation in synaptosomes, providing a rapid method for investigating early neuronal events that occur during oxidative stress.
在大鼠突触体中测量了对氧化刺激产生的氧反应性物种的形成。研究使用了非荧光探针2′,7′-二氯荧光素二乙酸酯（DCFH-DA），其在去酯化后在氧反应性物种存在下被氧化为高荧光的2′,7′-二氯荧光素（DCF）。通过DCF荧光测量的氧反应性物种形成受到抗坏血酸和/或硫酸亚铁（FeSO₄）以及黄嘌呤/黄嘌呤氧化酶在各种缓冲条件下的刺激。这些试剂均增加了Tris、HEPES和磷酸盐缓冲液中DCF的形成。抗坏血酸还以浓度依赖性方式刺激DCF的形成。HEPES缓冲液中Ca²⁺的存在并未增强或减弱抗坏血酸/硫酸亚铁对DCF形成的影响。去铁胺抑制了抗坏血酸/硫酸亚铁诱导的DCF形成刺激，但超氧化物歧化酶预处理对黄嘌呤/黄嘌呤氧化酶诱导的刺激没有影响。结果表明，DCF荧光是突触体中氧反应性物种形成的一种灵敏、定量且直接的测量方法，为研究氧化应激期间发生的早期神经元事件提供了一种快速方法。
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