Wang Hsing-Wen, Gukassyan Vladimir, Chen Chien-Tsun, Wei Yau-Huei, Guo Han-Wen, Yu Jia-Sin, Kao Fu-Jen
National Yang-Ming University, Institute of Biophotonics, Taipei, Taiwan.
J Biomed Opt. 2008 Sep-Oct;13(5):054011. doi: 10.1117/1.2975831.
Direct monitoring of cell death (i.e., apoptosis and necrosis) during or shortly after treatment is desirable in all cancer therapies to determine the outcome. Further differentiation of apoptosis from necrosis is crucial to optimize apoptosis-favored treatment protocols. We investigated the potential modality of using tissue intrinsic fluorescence chromophore, reduced nicotinamide adenine dinucleotide (NADH), for cell death detection. We imaged the fluorescence lifetime changes of NADH before and after staurosporine (STS)-induced mitochondria-mediated apoptosis and hydrogen peroxide (H2O2)-induced necrosis, respectively, using two-photon fluorescence lifetime imaging in live HeLa cells and 143B osteosarcoma. Time-lapsed lifetime images were acquired at the same site of cells. In untreated cells, the average lifetime of NADH fluorescence was approximately 1.3 ns. The NADH average fluorescence lifetime increased to approximately 3.5 ns within 15 min after 1 microM STS treatment and gradually decreased thereafter. The NADH fluorescence intensity increased within 15 min. In contrast, no significant dynamic lifetime change was found in cells treated with 1 mM H2O2. Our findings suggest that monitoring the NADH fluorescence lifetime may be a valuable noninvasive tool to detect apoptosis and distinguish apoptosis from necrosis for the optimization of apoptosis-favored treatment protocols and other clinical applications.
在所有癌症治疗过程中或治疗后不久直接监测细胞死亡（即凋亡和坏死）对于确定治疗结果是很有必要的。进一步区分凋亡和坏死对于优化倾向于凋亡的治疗方案至关重要。我们研究了利用组织固有荧光发色团——还原型烟酰胺腺嘌呤二核苷酸（NADH）进行细胞死亡检测的潜在方式。我们分别在活的HeLa细胞和143B骨肉瘤细胞中，使用双光子荧光寿命成像技术，对星形孢菌素（STS）诱导的线粒体介导的凋亡以及过氧化氢（H₂O₂）诱导的坏死前后NADH的荧光寿命变化进行成像。在细胞的同一部位采集了时间分辨的寿命图像。在未处理的细胞中，NADH荧光的平均寿命约为1.3纳秒。用1微摩尔STS处理后15分钟内，NADH的平均荧光寿命增加到约3.5纳秒，此后逐渐下降。NADH荧光强度在15分钟内增加。相比之下，用1毫摩尔H₂O₂处理的细胞未发现明显的动态寿命变化。我们的研究结果表明，监测NADH荧光寿命可能是一种有价值的非侵入性工具，用于检测凋亡并区分凋亡与坏死，以优化倾向于凋亡的治疗方案及其他临床应用。
