| Summary: | 博士 === 國立陽明大學 === 生醫光電研究所 === 101 === Reduced nicotinamide adenine dinucleotide (NADH) is an important coenzyme involved in numerous biochemical reactions in the cell. Particularly, it is a main proton (H+) carrier in cellular metabolism. In addition, NADH is a wildly used intrinsic biomarker for monitoring metabolic status, detecting and diagnosing cancer in past decades. In pervious studies, we observed that NADH fluorescence and lifetime increase in the beginning of staurosporine-induced apoptosis that differed to the change of NADH fluorescence due to metabolism change. In this thesis, we investigate the role of intrinsic optical biomarker, NADH fluorescence, in cell death and metabolism models by PARP-1 induced cell death and human mesenchymal stem cell differentiation, respectively. We also measure mitochondrial function to delineate the relationship between mitochondrial activity and NADH fluorescence.
In this study, we induced PARP-1 hyperactivation by treating cells with methyl-nitro-nitroso-guanidine (MNNG). During PARP-1 hyperactivation, oxidized nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) were depleted to process poly-ADP ribosylation, which caused the inhibition of respiration and cell death. In this case, we found that NADH fluorescence intensity decreases rapidly, NADH fluorescence lifetime increase, and mitochondrial dysfunction during PARP-1 induced cell death. Furthermore, upon the treatment of pyruvate to rescue the cells, we found that the increase of NADH fluorescence lifetime was slowed down and mitochondrial function was maintained. These findings indicate that NADH fluorescence lifetime can reflect the severity of PARP-1-induced cell death.
In stem cell differentiation, there is a metabolic shift from anaerobic glycolysis to more effective oxidative phosphorylation. We found that NADH fluorescence lifetime of human mesenchymal stem cell (hMSCs) increased gradually along the course of osteogenic differentiation. The metabolic shift also occurred during osteogenic differentiation and the change of NADH fluorescence lifetime correlated well with the ATP level and oxygen consumption rate. These results indicate that NADH fluorescence lifetime can reflect metabolic change of stem cell differentiation. In addition, we found that the expression of respiratory enzymes increased after osteogenic induction. We suggest that the increase in the expression of respiratory enzymes contributed to the increase of NADH fluorescence lifetime.
In this study, we demonstrated that NADH fluorescence lifetime is a potential noninvasive biomarker to diagnose the diseases with PARP-1-induced cell death and detect the “stemness” or differentiated level of stem cells. Based on this and previous studies, we also suggest that the role of NADH fluorescence lifetime is different in cell death and normal metabolic changes due to its interaction with different molecules, which leads to the variable increase of NADH fluorescence time.
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