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目的研究实验动物心肌线粒体氧化还原状态,监测缺血时线粒体功能改变的早期信号。方法线粒体呼吸链主要电子供应者烟酰胺腺嘌呤(磷酸)二核苷酸,或NAD(P)H荧光为无创荧光标记,用光谱分辨的时间相关单光子计数(TCSPC)检测紫外光(UV)激发的心肌自发荧光(AF)光谱和荧光寿命。结果需用至少3个荧光寿命池0.4~0.7 ns,1.2~1.9 ns和8.0~13.0 ns描述心肌AF。相关衰减光谱(DAS)显示4个NAD(P)H荧光固有光谱,分别为峰值470 nm的短荧光寿命池,及450 nm,470 nm和490 nm的中间和长荧光寿命池。酮体增加线粒体NADH产量,提高AF强度,但不改变荧光寿命。线粒体呼吸阻断剂Rotenone,显著增加AF强度和缩短平均荧光寿命。氧化磷酸化解偶联剂Dinitrophenol(DNP),显著降低AF强度,在520 nm处增宽荧光光谱并显著延长平均荧光寿命。这些结果和NADH荧光动力学离体实验(in vitro)及模拟缺血状态实验有可比性。结论研究可解释NADH构象改变,以及从NADH到异戎酸脱氢酶(LipDH)结合的黄素蛋白间能量转移的荧光动力学变化。光谱分辨的荧光寿命显微技术提供了在细胞水平上研究心肌能量代谢或线粒体功能障碍的新工具。
OBJECTIVE: To study the mitochondrial redox status of experimental animals and to monitor early signals of mitochondrial function changes during ischemia. Methods The main electron donor of mitochondrial respiratory chain was nicotinamide adenine (phosphoric acid) dinucleotide, or NAD (P) H fluorescence was used as noninvasive fluorescent marker to detect ultraviolet (UV) light with time-dependent single photon counting (TCSPC) Excited myocardial autofluorescence (AF) spectra and fluorescence lifetime. Results Describe myocardial AF with at least 3 fluorescence lifetime pools of 0.4 to 0.7 ns, 1.2 to 1.9 ns, and 8.0 to 13.0 ns. Correlated Attenuation Spectroscopy (DAS) revealed four NAD (P) H fluorescence intrinsic spectra, short fluorescence lifetime peak at 470 nm, and intermediate and long fluorescence lifetime pools at 450 nm, 470 nm and 490 nm, respectively. Ketone increases mitochondrial NADH production, increases AF intensity but does not change fluorescence lifetime. Rotenone, a mitochondrial respiratory blocker, significantly increases AF intensity and shortens the average fluorescence lifetime. Dinitrophenol (DNP), an oxidative phosphorylation uncoupler, significantly reduced AF intensity, broadened the fluorescence spectrum at 520 nm and significantly extended the mean fluorescence lifetime. These results are comparable to NADH fluorescence kinetics in vitro and simulated ischemic state experiments. Conclusions The study may explain the change in conformation of NADH as well as the change in fluorescence kinetics of energy transfer between flavonins bound by NADH and isodonate dehydrogenase (LipDH). Spectrally resolved fluorescence lifetime microscopy provides a new tool to study myocardial energy metabolism or mitochondrial dysfunction at the cellular level.