Voogt Jason N, Awada Mohamad, Murphy Elizabeth J, Hayes Gregory M, Busch Robert, Hellerstein Marc K
KineMed Inc., 5980 Horton Street, Emeryville, California 94608, USA.
Nat Protoc. 2007;2(12):3058-62. doi: 10.1038/nprot.2007.421.
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.
S期的DNA复制代表了细胞分裂的一种生化指标。我们在此介绍一种测量极低细胞增殖率的方法，该方法基于重水（²H₂O）中的氘（²H）掺入分裂细胞DNA中嘌呤脱氧核糖核苷酸的脱氧核糖部分，采用气相色谱/热解/同位素比率质谱法（GC/P/IRMS）。通过GC/P/IRMS可以对极低水平的标记掺入（≥0.002%原子百分超量²H）进行定量。因此，与使用标准GC/MS技术测量细胞增殖动力学相比，该方法允许更短的²H₂O给药时间或更低的²H₂O给药量（见本期附带的方法）。与GC/MS相比，这种方法的一个缺点是需要更多的细胞（>约10⁷个）。该方法能够对动物和人类中新分裂细胞的比例或绝对数量及其随后的存活动力学进行明确的体内研究，即使周转率非常低。惰性血液系统恶性肿瘤，如慢性淋巴细胞白血病，以及其他相对静止的细胞，是很有前景的应用领域。
