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目的:应用心肌自发荧光(AF)研究心肌线粒体氧化代谢状态,监测线粒体功能改变的早期信号。方法:烟酰胺腺嘌呤(磷酸)二核苷酸[NAD(P)H]作为荧光探针,用光谱分辨的时间相关单光子计数(TCSPC)记录375 nm紫外激光激发的心肌AF光谱和荧光寿命,测试影响线粒体呼吸时AF动态衰减。结果:在420~560 nm光谱区域,至少需用3个荧光寿命池0.4~0.7 ns,1.2~1.9 ns和8.0~13.0 ns描述细胞AF。线粒体呼吸阻断剂鱼藤酮可显著增加AF强度,缩短平均荧光寿命。氧化磷酸化解偶联剂二硝基酚可显著降低AF强度,在520 nm处增宽荧光光谱,延长平均荧光寿命。这些结果和NADH荧光动力学离体实验(in vitro)有可比性。结论:光谱分辨的荧光寿命技术测定心肌NAD(P)H荧光有很好的重复性,在细胞水平上增加了心肌氧化代谢或线粒体功能障碍的知识,为临床诊断和治疗线粒体功能障碍开拓了新视野。
OBJECTIVE: To study the oxidative metabolism of myocardial mitochondria by autofluorescence (AF) and to detect early signals of mitochondrial function changes. METHODS: Nicotinamide adenine (phosphoric acid) dinucleotide [NAD (P) H] was used as a fluorescent probe to record the AF spectrum and fluorescence lifetime of 375 nm UV laser-activated myocardium using a spectrally resolved time-correlated single photon counting (TCSPC) , Testing the dynamic attenuation of mitochondrial AF affecting mitochondria. Results: In the 420-560 nm spectral region, at least 3 fluorescence lifetime pools, 0.4 to 0.7 ns, 1.2 to 1.9 ns, and 8.0 to 13.0 ns, are required to describe the cellular AF. Rotenone, a mitochondrial respiratory blocker, significantly increases AF intensity and shortens the mean fluorescence lifetime. Oxidative phosphorylation of uncoupler dinitrophenol can significantly reduce the AF intensity, broadening the fluorescence spectrum at 520 nm to extend the average fluorescence lifetime. These results are comparable to NADH fluorescence in vitro experiments. CONCLUSIONS: Spectral-resolved fluorescence lifetime assay of myocardial NAD (P) H fluorescence shows good reproducibility and increases cellular knowledge of myocardial oxidative metabolism or mitochondrial dysfunction at the cellular level, opening up new possibilities for the clinical diagnosis and treatment of mitochondrial dysfunction Vision.