Quantifying cell division with deuterated water and multi-isotope imaging mass spectrometry (MIMS).
Quantifying cell division with deuterated water and multi-isotope imaging mass spectrometry (MIMS).
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DOI：
10.1002/sia.5581
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发表时间：
2014-11-01
期刊：
影响因子：
--
通讯作者：
Lechene CP
中科院分区：
文献类型：
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作者：
Steinhauser ML;Guillermier C;Wang M;Lechene CP
细胞分裂通常通过施用通过核苷酸补救途径掺入的核苷酸标记来定量。一种新的方法使用从头核苷酸合成途径的前体，如标记水或葡萄糖。由于这些前体对DNA合成不是特异性的，因此利用这种方法的研究已经分析了分离的基因组DNA以排除非特异性背景标记。我们假设，脉冲追逐管理的稳定同位素标记的水将导致足够的核标记，使最近分裂的细胞的定量离子显微镜的歧视。我们同时给予小鼠氘代（D）水和15 N-胸苷，指导的基本原理是15 N-胸苷掺入将作为“金标准”，以确定分裂细胞。我们定性和定量地表明，分裂细胞在小肠（15 N-标记）表现出可辨别的D-信号在细胞核中未观察到未分裂的细胞（15 N-未标记）。与31 P −和12 C15 N −：12 C14 N −图像的相关性表明，2 H标记优先定位在具有高DNA含量的细胞核区域，正如预期的那样，在DNA合成和细胞分裂期间标记被掺入。这些数据支持这样的概念，即从头核苷酸合成途径的稳定同位素标记的前体可以与NanoSIMS一起用于研究体内细胞分裂。这项研究的一个主要意义是使用稳定同位素标记水和MIMS研究人类细胞更新的可能性。
Cell division is commonly quantified by the administration of nucleotide labels that are incorporated by the nucleotide salvage pathway. A new approach uses precursors of the de novo nucleotide synthesis pathway, such as labeled water or glucose. Because such precursors are not specific for DNA synthesis, studies utilizing this approach have analyzed isolated genomic DNA to exclude nonspecific background labeling. We hypothesized that pulse-chase administration of stable isotope labeled water would result in sufficient nuclear labeling to enable discrimination of recently divided cells by quantitative ion microscopy. We administered deuterated (D)-water and 15N-thymidine to mice concurrently, guided by the rationale that 15N-thymidine incorporation would serve as a “gold standard” to identify dividing cells. We show both qualitatively and quantitatively that dividing cells in the small intestine (15N-labeled) demonstrate a discernable D-signal in the nucleus not observed in undivided cells (15N-unlabled). Correlation with 31P− and 12C15N−:12C14N− images demonstrate preferential localization of 2H labeling in regions of the nucleus with high DNA content as expected of labeling being incorporated during DNA synthesis and cell division. These data support the concept that stable isotope tagged precursors of the de novo nucleotide synthesis pathway can be used in concert with NanoSIMS to study cell division in vivo. A major implication of this study then is the possibility of using stable isotope tagged water and MIMS to study human cell turnover.