A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes
A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes
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DOI：
10.1096/fj.04-2729fje
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发表时间：
2004-12-01
期刊：
影响因子：
4.8
通讯作者：
Ames, BN
中科院分区：
文献类型：
--
作者：
Offer, T;Ho, E;Ames, BN
暴露于环境压力，如辐射，营养不良或吸烟，可能会导致DNA的有害损伤，包括双链断裂。在红细胞中，在红细胞生成过程中，主核被挤出形成成熟网织红细胞后，DNA片段或落后染色体在留下时形成微核。人外周血中含微核的网织红细胞通常不可用于分析，因为脾脏可清除异常细胞。我们已经开发了一种简单快速的方法来分离和分析外周血中未成熟的网织红细胞，在这些细胞被脾脏去除之前，这些细胞是否存在微核。该方法应用单激光流式细胞术测量富集转铁蛋白阳性网织红细胞群体中的微核。Abramsson-Zetterberg等人（Abramsson-Zetterberg，L.，Zetterberg，G.，Bergqvist，M.，和Grawe，J. Environ。摩尔变异体36，22 - 31，2000）已经描述了使用双激光流式细胞术测量富集的网织红细胞群体中的微核的方法。与其磁性免疫分离程序中使用的珠粒不同，本研究中使用的珠粒不需要预标记步骤，并且与流动池相容，从而无需从珠粒中释放细胞并避免潜在的混淆DNA酶处理步骤。Dertinger等人（Dertinger，S. D、Torous，D. K.，霍尔，N。E、Murante，F. G.，格里森，S。E、米勒河K.，和Tometsko，C. R.穆塔Res. 515，3 - 14，2002; Dertinger，S. D、陈玉，米勒河K.，Brewer，K. J.，Smudzin，T.，Torous，D. K.，霍尔，N。E、奥尔瓦尼湾一、Murante，F. G.，和Tometsko，C. R.（2003）Mutat. Res. 542，77 - 87，2003）进一步改进了微核的评分，使得能够使用台式仪器分析未富集的网织红细胞群体的样品。本方法不同于流式细胞术测定，如Dertinger等人报道的，这使得每个样本能够对有限数量的网织红细胞进行评分，并且需要很长的数据采集时间。我们使用这种新的基于流式细胞术的微核试验评估吸烟者的DNA损伤。结果表明，该方法可有效地检测人体血液样品中的微核。与现有的微核试验不同，该方法可以快速评价大量细胞，因此应证明可用于监测人群的遗传损伤。
Exposure to environmental stress, such as radiation, poor nutrition, or smoking, can cause hazardous lesions in DNA, including double-strand breaks. In red blood cells, a DNA fragment or lagging chromosome forms a micronucleus when left behind after the main nucleus is extruded to form the mature reticulocyte during erythropoiesis. Reticulocytes with micronuclei in human peripheral blood are not generally available for analysis because the spleen removes aberrant cells. We have developed a simple and rapid method to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei before these cells are removed by the spleen. This method applies single-laser flow-cytometry to measure micronuclei in an enriched transferrin-positive reticulocyte population. Abramsson-Zetterberg et al. (Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M., and Grawe, J. Environ. Mol. Mutagen. 36, 22 - 31, 2000) have described a method to measure micronuclei in an enriched reticulocyte population using a dual-laser flow cytometry. Unlike the beads used in their magnetic-immunoseparation procedures, the beads used in this study do not require a prelabeling step and are compatible with the flow cell, sparing the need to release the cells from the beads and avoiding the potentially confounding DNase-treatment step. Dertinger et al. ( Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G., Gleason, S. E., Miller, R. K., and Tometsko, C. R. Mutat. Res. 515, 3 - 14, 2002; Dertinger, S. D., Chen, Y., Miller, R. K., Brewer, K. J., Smudzin, T., Torous, D. K., Hall, N. E., Olvany, K. A., Murante, F. G., and Tometsko, C. R. ( 2003) Mutat. Res. 542, 77 - 87, 2003) further improved the scoring of micronuclei to enable the use of benchtop instruments in analyzing samples of unenriched reticulocyte-populations. The present method is distinct from flow cytometric assays, such as reported by Dertinger et al., which enable scoring of limited numbers of reticulocytes per sample and require lengthy data acquisition times. We assessed DNA damage in smokers using this novel flow-cytometry based micronuclei-assay. The results show that this assay can effectively detect micronuclei in human blood samples. This method, unlike available micronuclei assays, allows rapid evaluation of a large number of cells and therefore should prove to be useful in monitoring of human populations for genetic damage.