Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka, Japan.
J Toxicol Sci. 2011 Aug;36(4):435-44. doi: 10.2131/jts.36.435.
To evaluate the effects of a variety of chemical, biological and physiological stimuli on erythropoiesis, in vitro assays using erythroid progenitor cells from humans or laboratory animals are well-known methods. On the other hand, little has been reported on in vitro assays using mature erythroblasts such as polychromatic erythroblasts. In the present study, we established a convenient method for enrichment of polychromatic erythroblasts from rat bone marrow and confirmed their development in vitro. To establish a method for the enrichment of polychromatic erythroblasts, bone marrow cells from 3- and 10-week-old rats were separated by discontinuous density gradient centrifugation using Percoll. As a result, polychromatic erythroblasts were most highly enriched in the bone marrow fraction from 3-week old rats at the density interface between 1.040 and 1.058 g/ml. The enriched polychromatic erythroblasts were then cultured in growth medium supplemented with 20% fetal bovine serum in the presence or absence of erythropoietin for 48 hr. During the culture period, cell proliferation and maturation to orthochromatic erythroblasts were observed, and intracellular heme contents were also increased. In particular, the culture in the presence of erythropoietin revealed higher proliferation of erythroid cells, and therefore might be more appropriate for in vitro experiments on the effects of various stimuli on late-stage erythropoiesis.
为了评估各种化学、生物和生理刺激对红细胞生成的影响，使用来自人类或实验动物的红系祖细胞的体外检测方法是众所周知的方法。另一方面，关于使用成熟的红细胞（如多染红细胞）的体外检测方法报道甚少。在本研究中，我们建立了一种从大鼠骨髓中富集多染红细胞的简便方法，并证实了它们在体外的发育。为了建立一种富集多染红细胞的方法，使用 Percoll 通过不连续密度梯度离心从 3 周和 10 周龄大鼠的骨髓细胞中分离。结果，在 1.040 和 1.058 g/ml 之间的密度界面处，3 周龄大鼠骨髓中的多染红细胞得到了最高程度的富集。然后，将富集的多染红细胞在添加有 20%胎牛血清的生长培养基中，在存在或不存在促红细胞生成素的情况下培养 48 小时。在培养期间，观察到细胞增殖和向正染红细胞的成熟，并且细胞内血红素含量也增加。特别是，在促红细胞生成素存在的情况下进行培养显示出红细胞的更高增殖，因此可能更适合于研究各种刺激对晚期红细胞生成的影响的体外实验。
