Sawada K, Krantz S B, Dai C H, Koury S T, Horn S T, Glick A D, Civin C I
Department of Medicine, Veterans Administration Medical Center, Nashville, Tennessee.
J Cell Physiol. 1990 Feb;142(2):219-30. doi: 10.1002/jcp.1041420202.
To facilitate the direct study of the molecular events that control the development of human burst-forming units-erythroid (BFU-E), we have developed a method to purify BFU-E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase-treated and IgG-coated sheep erythrocytes, positive panning with anti-My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU-E were purified from 0.04% to 56.6%, a 1,400-fold purification with a 13% yield. More than 90% of purified BFU-E were recombinant interleukin-3 (rIL-3) dependent, which survived for 48 h with rIL-3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rIL-3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony-forming units-erythroid (CFU-E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture. 125I-rEP was incubated for 5 h at 37 degrees C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24-72 h, and the presence of erythropoietin (EP) receptors was investigated using autoradiography. Specific binding of 125I-rEP was detected in 19 +/- 7% of the initial day 1 BFU-E. The percentage of 125I-rEP-binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU-E matured. 125I-rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU-E have a much lower number of EP receptors than CFU-E and develop an increased concentration of EP receptors in association with their maturation and loss of proliferative capacity.
为便于直接研究控制人类红系爆式集落形成单位（BFU-E）发育的分子事件，我们开发了一种从外周血中纯化BFU-E的方法。通过密度离心、用神经氨酸酶处理过的和IgG包被的绵羊红细胞混合物进行玫瑰花结形成、用抗My10单克隆抗体进行阳性淘选、在塑料培养皿上过夜贴壁、用单克隆抗体进行阴性淘选以及密度离心，人类血液中的BFU-E从0.04%纯化至56.6%，纯化倍数达1400倍，产率为13%。超过90%的纯化BFU-E依赖重组白细胞介素-3（rIL-3），在无重组促红细胞生成素（rEP）的情况下，rIL-3可使其存活48小时，且80%能产生超过500个血红蛋白化细胞的红系集落。直到体外培养72小时后，rEP依赖性才明显显现。将纯化的细胞（第1天）在液体培养中与rIL-3和rEP一起培养24小时（第2天）、48小时（第3天）和72小时（第4天），然后转移至半固体培养中并培养至第15天。在半固体培养中观察到的红系集落大小与第1天纯化细胞在液体培养中的孵育时间相关，持续减小。在第1天细胞于液体培养中孵育72小时后，首次观察到产生8至49个细胞集落的红系集落形成单位（CFU-E）。将125I-rEP与纯化细胞（第1天）或在液体培养中再额外培养24 - 72小时的细胞在37℃孵育5小时，然后用放射自显影法研究促红细胞生成素（EP）受体的存在情况。在初始第1天的BFU-E中，19±7%检测到125I-rEP的特异性结合。随着第1天BFU-E的成熟，125I-rEP与红系祖细胞结合的百分比及结合量持续增加。在液体培养中孵育72小时的所有红系祖细胞中均观察到125I-rEP特异性结合。这些数据表明，原始的BFU-E比CFU-E具有少得多的EP受体，并且随着其成熟和增殖能力的丧失，会形成浓度增加的EP受体。
