光谱分辨的NAD(P)H荧光寿命光谱学对心肌氧化代谢的评估  

Assessment of Mitochondrial Metabolic Oxidative State in Living Cardiomyocytes with Spectrally-Resolved Fluorescence Lifetime Spectroscopy of NAD(P)H

作　　者：程颖[1,2] 任明明[1] 牛延艳[1] 乔建华[1] Aneba S. Chorvat Jr.D. Chorvatova A. 

机构地区：[1]北京大学深圳医院心血管外科,深圳518036 [2]蒙特利尔大学儿科学部,蒙特利尔大学圣-贾斯汀医院研究中心 [3]斯洛伐克国际激光中心生物光子学部,伯拉第斯拉瓦斯洛伐克81219

出　　处：《生物医学工程学杂志》2009年第6期1191-1200,共10页Journal of Biomedical Engineering

基　　金：加拿大卫生研究院基金资助项目(MOP74600);加拿大创新基金资助项目(No9684);魁北克卫生研究基金资助项目(No2948)~~

摘　　要：应用光谱分辨的NAD(P)H荧光寿命光谱学研究心肌细胞线粒体氧化代谢状态,监测病理情况下线粒体功能改变的早期信号。研究将NAD(P)H作为无创荧光探针,用光谱分辨的时间相关单光子计数记录375nm紫外光激发的心肌自发荧光(AF)光谱和寿命,测试影响NADH生产或线粒体呼吸时AF动态衰减。结果显示:在420～560nm光谱区域,需用至少3个荧光寿命池0.4～0.7ns、1.2～1.9ns和8.0～13.0ns描述细胞AF。酮体增加NADH水平,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,缩短平均荧光寿命。氧化磷酸化解偶联剂(DNP),显著降低AF强度,在520nm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(invitro)有可比性。研究证实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,增加了心肌氧化代谢或线粒体功能障碍在细胞水平上的知识,为临床诊断和治疗线粒体功能障碍开拓了新视野。The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD（P）H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD（P） H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting （TCSPC）, based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD（P） H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9as and 8.0-13.0ns lifetime pools is necessary to describe eardiomyocyte autofluorescence （AF） within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that speetrally-resolved fluorescence lifetime technique provides promising new tool for analysis of m

关 键 词：烟酰胺腺嘌呤(磷酸)二核苷酸 自发荧光 光谱分辨的荧光寿命 线粒体 存活心肌细胞 

分 类 号：R318.51[医药卫生—生物医学工程] R329.27[医药卫生—基础医学]