• 1.北京大學(xué)深圳醫(yī)院心血管外科,中國深圳 518036;2.蒙特利爾大學(xué)兒科學(xué)部,蒙特利爾大學(xué)圣賈斯汀醫(yī)院研究中心,加拿大蒙特利爾 H3T 1C5;3.斯洛伐克國際激光中心生物光子學(xué)部,斯洛伐克伯拉第斯拉瓦 81219;

目的:應(yīng)用心肌自發(fā)熒光(AF)研究心肌線粒體氧化代謝狀態(tài),監(jiān)測線粒體功能改變的早期信號。方法:煙酰胺腺嘌呤(磷酸)二核苷酸［NAD(P)H］作為熒光探針,用光譜分辨的時間相關(guān)單光子計數(shù)(TCSPC)記錄375nm紫外激光激發(fā)的心肌AF光譜和熒光壽命,測試影響線粒體呼吸時AF動態(tài)衰減。結(jié)果:在420~560nm光譜區(qū)域,至少需用3個熒光壽命池0.4~0.7ns,1.2~1.9ns和8.0~13.0ns描述細(xì)胞AF。線粒體呼吸阻斷劑魚藤酮可顯著增加AF強(qiáng)度,縮短平均熒光壽命。氧化磷酸化解偶聯(lián)劑二硝基酚可顯著降低AF強(qiáng)度,在520nm處增寬熒光光譜,延長平均熒光壽命。這些結(jié)果和NADH熒光動力學(xué)離體實驗(in vitro)有可比性。結(jié)論:光譜分辨的熒光壽命技術(shù)測定心肌NAD(P)H熒光有很好的重復(fù)性,在細(xì)胞水平上增加了心肌氧化代謝或線粒體功能障礙的知識,為臨床診斷和治療線粒體功能障礙開拓了新視野。

引用本文： 程穎,任明明,劉云奇,Aneba S.,Chorvat Jr. D.,Chorvatova A.. 應(yīng)用心肌自發(fā)熒光評估存活心肌線粒體氧化代謝狀態(tài). 華西醫(yī)學(xué), 2009, 24(7): 1767-1771. doi: 復(fù)制

1.
2. CHANCE B, SCHOENER B, OSHINO R, et al. Oxidationreduction ratio studies of mitochondria in freezetrapped samples. NADH and flavoprotein fluorescence signals[J]. J Biol Chem,1979,254(11):4764-4771..
3. BLINOVA K, CARROLL S, BOSE S, et al. Distribution of mitochondrial NADH fluorescence lifetimes: steadystate kinetics of matrix NADH interactions[J]. Biochemistry,2005,44(7):2585-2594..
4. ENG J, LYNCH R M, BALABAN R S. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes[J]. Biophys J,1989,55(4):621-630..
5. CHANCE B, THORELL B. Fluorescence measurements of mitochondrial pyridine nucleotide in aerobiosis and anaerobiosis[J]. Nature,1959,184:931-934..
6. BASSIENCAPSA V, FOURON J C, COMTE B, et al. Structural, functional and metabolic remodeling of rat left ventricular myocytes in normal and in sodiumsupplemented pregnancy[J]. Cardiovasc Res,2006,69(2):423-431..
7. CHORVAT D JR, CHORVATOVA A. Spectrally resolved timecorrelated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes[J]. Eur Biophys J,2006,36:73-83..
8. ANEBA S, CHENG Y, MATEASIK A, et al. Probing of cardiomyocyte metabolism by spectrally resolved lifetime detection of NAD(P)H fluorescence[J]. Comput Cardiol,2007,34:349-352..
9. VISHWASRAO H D, HEIKAL A A, KASISCHKE K A, et al. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy[J]. J Biol Chem,2005,280(26):25119-25126..
10. WAKITA M, NISHIMURA G, TAMURA M. Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ[J]. J Biochem (Tokyo),1995,118(6):1151-1160..
11. BRAUTIGAM C A, CHUANG J L, TOMCHICK D R, et al. Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of diseasecausing mutations[J]. J Mol Biol,2005,350(3):543-552..
12. ROMASHKO D N, MARBAN E, O’ROURKE B. Subcellular metabolic transients and mitochondrial redox waves in heart cells[J]. Proc Natl Acad Sci USA,1998,95(4):1618-1623..
13. CHORVAT D JR, BASSIENCAPSA V, CAGALINEC M, et al. Mitochondrial autofluorescence induced by visible light in single cardiac myocytes studied by spectrally resolved confocal microscopy[J]. Laser Physics,2004,14(2):220-230..
14. CHORVAT D JR, KIRCHNEROVA J, CAGALINEC M, et al. Spectral unmixing of flavin autofluorescence components in cardiac myocytes[J]. Biophys J,2005,89(6):L55-L57..
15. CHORVAT D, ELZWIEI F, BASSIENCAPSA V, et al. Assessment of lowIntensity fluorescence signals in living cardiac cells using timeresolved laser spectroscopy[J]. Comput Cardiol,2007,34:353-357..
16. SEKAR R B, PERIASAMY A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations[J]. J Cell Biol,2003,160(5):629-633..
17. CHENG Y, DAHDAH N, PORIER N, et al. Spectrally and timeresolved study of NADH autofluorescence in cardiac myocytes from human biopsies[M]. Proceedings of SPIE: the International Society for Optical Engineering,2007:6771(Advanced Photon Counting Techniques II): 677104-1-67710413..
  1. 1.
  2. 2. CHANCE B, SCHOENER B, OSHINO R, et al. Oxidationreduction ratio studies of mitochondria in freezetrapped samples. NADH and flavoprotein fluorescence signals[J]. J Biol Chem,1979,254(11):4764-4771..
  3. 3. BLINOVA K, CARROLL S, BOSE S, et al. Distribution of mitochondrial NADH fluorescence lifetimes: steadystate kinetics of matrix NADH interactions[J]. Biochemistry,2005,44(7):2585-2594..
  4. 4. ENG J, LYNCH R M, BALABAN R S. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes[J]. Biophys J,1989,55(4):621-630..
  5. 5. CHANCE B, THORELL B. Fluorescence measurements of mitochondrial pyridine nucleotide in aerobiosis and anaerobiosis[J]. Nature,1959,184:931-934..
  6. 6. BASSIENCAPSA V, FOURON J C, COMTE B, et al. Structural, functional and metabolic remodeling of rat left ventricular myocytes in normal and in sodiumsupplemented pregnancy[J]. Cardiovasc Res,2006,69(2):423-431..
  7. 7. CHORVAT D JR, CHORVATOVA A. Spectrally resolved timecorrelated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes[J]. Eur Biophys J,2006,36:73-83..
  8. 8. ANEBA S, CHENG Y, MATEASIK A, et al. Probing of cardiomyocyte metabolism by spectrally resolved lifetime detection of NAD(P)H fluorescence[J]. Comput Cardiol,2007,34:349-352..
  9. 9. VISHWASRAO H D, HEIKAL A A, KASISCHKE K A, et al. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy[J]. J Biol Chem,2005,280(26):25119-25126..
  10. 10. WAKITA M, NISHIMURA G, TAMURA M. Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ[J]. J Biochem (Tokyo),1995,118(6):1151-1160..
  11. 11. BRAUTIGAM C A, CHUANG J L, TOMCHICK D R, et al. Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of diseasecausing mutations[J]. J Mol Biol,2005,350(3):543-552..
  12. 12. ROMASHKO D N, MARBAN E, O’ROURKE B. Subcellular metabolic transients and mitochondrial redox waves in heart cells[J]. Proc Natl Acad Sci USA,1998,95(4):1618-1623..
  13. 13. CHORVAT D JR, BASSIENCAPSA V, CAGALINEC M, et al. Mitochondrial autofluorescence induced by visible light in single cardiac myocytes studied by spectrally resolved confocal microscopy[J]. Laser Physics,2004,14(2):220-230..
  14. 14. CHORVAT D JR, KIRCHNEROVA J, CAGALINEC M, et al. Spectral unmixing of flavin autofluorescence components in cardiac myocytes[J]. Biophys J,2005,89(6):L55-L57..
  15. 15. CHORVAT D, ELZWIEI F, BASSIENCAPSA V, et al. Assessment of lowIntensity fluorescence signals in living cardiac cells using timeresolved laser spectroscopy[J]. Comput Cardiol,2007,34:353-357..
  16. 16. SEKAR R B, PERIASAMY A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations[J]. J Cell Biol,2003,160(5):629-633..
  17. 17. CHENG Y, DAHDAH N, PORIER N, et al. Spectrally and timeresolved study of NADH autofluorescence in cardiac myocytes from human biopsies[M]. Proceedings of SPIE: the International Society for Optical Engineering,2007:6771(Advanced Photon Counting Techniques II): 677104-1-67710413..