心肌细胞NAD(P)H荧光成分分析法监测心脏移植排斥反应

Analysis of NAD(P)H Fluorescence Components in Cardiomyocyte to Detect Allograft Rejection in Heart Transplantation

目的为早期诊断和有效预防心脏移植排斥反应,研究在细胞水平用心肌NAD(P)H荧光成分分析监测排斥反应的可行性。方法用NAD(P)H荧光标记监测线粒体功能,从14例儿童心脏移植患者心内膜活检组织中分离活体心肌细胞和大鼠(n=5,13～14周龄)心肌细胞,光谱分辨的时间相关单光子计数(TCSPC)记录375nm紫外激光激发的心肌自发荧光(AF)光谱和荧光寿命。将鼠心肌细胞分为4组,无缺氧对照组,无缺氧鱼藤酮组,缺血对照组和缺血鱼藤酮组;对比无排斥反应(R0)人心肌细胞,无缺氧鼠细胞和缺血鼠细胞间,R0人心肌细胞和轻度排斥反应(R1)间的AF动力学变化。结果成功分离并测定了人心肌细胞AF光谱,在420～560nm光谱区域,至少存在0.5～0.7ns,1.9～2.4ns和9.0～15.0ns3个AF寿命池。与无缺氧对照组比较,缺血对照组鼠心肌细胞荧光强度明显增强(P<0.05);无缺氧鱼藤酮组和缺血鱼藤酮组心肌AF强度均显著增强,两组比较差异无统计学意义(P>0.05)。R0组人心肌细胞AF强度显著弱于无缺氧对照组鼠心肌细胞(P<0.05);人心肌AF强度与排斥反应相关,R1的荧光强度显著增强(P<0.05),但光谱形态无改变,与缺血对照组鼠心肌细胞光谱改变一致。结论光谱分辨的荧光光谱结合荧光寿命分析是监测心肌线粒体氧化代谢的敏感方法,有良好的重复性。人心肌AF表现出较高的代谢活性,排斥反应时活性降低。该技术结合多光子共聚焦显微镜,可直接在体评估线粒体功能,是监测早期排斥反应的新方法。

Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart,investigate possible applications of NAD（P）H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD（P）H was studied for non-invasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy （EMB） of 14 pediatric patients with heart transplantation. Rat cardiomyocyte （n=5,13-14 week old） were also isolated by the same approach for human myocytes. Autofluorescence（AF） was recorded in living cardiomyocytes following excitation with 375 nm UV-light and detection by spectrally-resolved time correlated single photon counting （TCSPC）,based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups：normoxic condition,normoxia with Rotenone,ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemia-mimicking ones,induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection （R0） or mild rejection （R1）. Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies,as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns,1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420-560 nm spectral range. Rat cardiomyocyte steady-state AF in ischemia-mimicking condition was significantly increased when compared normoxic ones （P〈0.05）; application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition,however no significant difference between the two groups （P〈0.05）.Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition（P〈0.05）. A correlation between changes in steady-state NAD（P）H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity （P〈0.05）,without change in the spectra shape,results can be comparable to the effect of ischemia-mimic conditions. Conclusion Our studies clearly demonstrated that spectrally-resolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones,while this activity （and thus ATP production） seems lowered during rejection process. In perspective,the advantage of this method is the possibility of its combination to multiphoton confocal microscopy,which can result in the adaptation of this approach directly to tissue biopsy,as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection,and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.