Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH
Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH
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DOI：
10.1158/0008-5472.can-04-3922
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发表时间：
2005-10-01
期刊：
影响因子：
11.2
通讯作者：
Ramanujam, N
中科院分区：
文献类型：
--
作者：
Bird, DK;Yan, L;Ramanujam, N
NADH浓度的生物化学估计是用于监测细胞代谢的有用方法，因为NADH/NAD（+）还原-氧化对对于线粒体电子链中的电子传递至关重要。在这篇文章中，我们提出了一种新的方法来推导功能地图的细胞内还原-氧化比在体内通过测量的荧光寿命和比率的自由和蛋白结合的NADH使用双光子荧光寿命成像（FLIM）。通过对来自对照细胞的FLIM数据的系统分析，观察到随着细胞从早期发展到对数期再到汇合期，游离和蛋白结合的NADH的荧光寿命以及蛋白结合的NADH的贡献在统计学上显著降低。氰化钾（KCN）处理和血清饥饿的细胞产生类似的变化。在生长曲线的早期和对数期，蛋白结合和游离NADH的荧光寿命有统计学显著性降低，并且在生长曲线的所有三个阶段，相对于对照细胞中观察到的蛋白结合NADH的贡献有统计学显著性降低。预期所施加的扰动（汇合、血清饥饿和KCN处理）均会导致NADH/NAD（+）比率增加。我们的研究表明，游离和蛋白结合的NADH组分的荧光寿命以及游离与蛋白结合的NADH的比率与NADH/NAD（+）比率的变化有关。
Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD(+) reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of protein-bound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD(+). Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD(+) ratio.