Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose
Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose
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DOI：
10.1038/nprot.2009.117
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Beverley, Peter C. L.
中科院分区：
文献类型：
--
作者：
Macallan, Derek C.;Asquith, Becca;Beverley, Peter C. L.
细胞增殖可以通过用同位素标记的脱氧核糖核苷酸前体定量DNA合成来体内测量。氘标记的葡萄糖是这样一种前体，因为它在短时间内实现高水平的富集，非常适合于快速分裂细胞的研究，与重水（（H2O）-H-2）实现的长期标记相反。由于氘是非放射性的，并且可以容易地施用葡萄糖，因此这种方法适合于临床研究。它已被广泛应用于研究人类淋巴细胞增殖，但也可以分析固体组织样品。[6，6-H-2（2）]-葡萄糖给药的速率、持续时间和途径（静脉或口服）应适应目标靶细胞。对于淋巴细胞，细胞分离最好通过荧光激活细胞分选（FACS）实现，尽管磁珠分离是一种替代方法。然后提取DNA，酶促水解并通过气相色谱质谱法（GC/MS）分析。适当的数学模型对解释至关重要。典型的时间要求如下：标记，10-24 h;取样，类似于3周; DNA提取/衍生化，2-3 d; GC/MS分析，类似于2 d。
Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water ((H2O)-H-2). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-H-2(2)]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10-24 h; sampling, similar to 3 weeks; DNA extraction/derivatization, 2-3 d; and GC/MS analysis, similar to 2 d.