Jones R J, Barber J P, Vala M S, Collector M I, Kaufmann S H, Ludeman S M, Colvin O M, Hilton J
Johns Hopkins Oncology Center, Johns Hopkins Medical Institutions, Baltimore, MD 21287-8967, USA.
Blood. 1995 May 15;85(10):2742-6.
Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.
胞质醛脱氢酶（ALDH）是一种负责氧化细胞内醛类的酶，在乙醇、维生素A和环磷酰胺代谢中起重要作用。该酶在包括骨髓和肠道在内的多种组织的原始干细胞中高表达，似乎是这些细胞对环磷酰胺耐药的重要机制。然而，尽管造血干细胞（HSC）表达高水平的胞质ALDH，但由于ALDH是一种细胞内蛋白，因此无法通过其ALDH表达来分离活的HSC。我们发现，一种荧光醛——丹磺酰氨基乙醛（DAAA），可用于流式细胞术实验，以根据细胞的ALDH含量分离活的小鼠和人类细胞。细胞与DAAA孵育后呈现的丹磺酰荧光水平与通过蛋白质免疫印迹法测定的胞质ALDH水平以及细胞对环磷酰胺的敏感性平行。此外，DAAA似乎是一种比蛋白质免疫印迹法更敏感的评估胞质ALDH水平的方法。用DAAA处理的骨髓祖细胞正常增殖。此外，与DAAA孵育后表达高水平丹磺酰荧光的骨髓细胞富含造血祖细胞。分离表达高水平胞质ALDH的活细胞的能力可能是鉴定和纯化HSC以及研究耐环磷酰胺肿瘤细胞群体方法的重要组成部分。
