Macallan D C, Fullerton C A, Neese R A, Haddock K, Park S S, Hellerstein M K
Department of Nutritional Sciences, University of California at Berkeley 94720, USA.
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):708-13. doi: 10.1073/pnas.95.2.708.
A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.
本文介绍了一种在体内测量DNA合成从而测定细胞增殖的方法。该技术包括给予[6,6-2H2]葡萄糖或[U-13C]葡萄糖，分离基因组DNA，酶解以释放脱氧核糖核苷，并进行衍生化用于气相色谱-质谱联用分析dA或dG的同位素富集情况，或两者皆分析。通过应用前体-产物关系，将dA或dG与细胞外葡萄糖富集情况进行比较（校正细胞内稀释），可揭示新合成DNA的比例。因此，该技术与广泛使用的[3H]胸腺嘧啶核苷或溴脱氧尿苷技术不同，在于它利用从头核苷酸合成途径而非核苷补救途径来标记DNA；标记的是脱氧核糖而非碱基部分；分析的是嘌呤而非嘧啶脱氧核糖核苷；并且使用的是稳定同位素而非放射性同位素。该方法在体外应用于培养的HepG2和H9细胞的生长；在动物中应用于肠上皮、胸腺和肝脏的增殖；在人体中应用于血液中粒细胞的更新。在所有情况下，测得的细胞增殖动力学与预期或独立测得的动力学一致。该方法相对于先前用于测量细胞更新的技术具有多个优点，不涉及放射性或潜在有毒代谢物，且适用于人体。一种用于测量人体细胞增殖的可靠且安全的方法的出现，开启了许多有待直接实验验证的基本问题，包括与癌症、艾滋病和其他病理状态相关的基础问题。
