Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells.
Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells.
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DOI：
10.1182/blood.v80.8.1940.bloodjournal8081940
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发表时间：
1992-10
期刊：
影响因子：
20.3
通讯作者：
A. Wickrema;S. Krantz;J. Winkelmann;M. Bondurant
中科院分区：
文献类型：
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作者：
A. Wickrema;S. Krantz;J. Winkelmann;M. Bondurant
从外周血中分离培养部分纯化的人红系(BFU-E)细胞，培养6~8d，获得集落形成单位(CFU-E)细胞。BFU-E来源的CFU-E进一步纯化后，在含有促红细胞生成素(EPO)的液体悬浮培养中重新培养，它们在体外成熟并分化为网织红细胞。通过测量~(59)Fe对血红素的掺入，在累积培养时间的第10天观察到最大的血红蛋白合成速率。在发育过程中的不同时间从红细胞培养物中撤除EPO表明，在第10天至第11天(大多数细胞处于多染红细胞阶段)，这些细胞变得独立于EPO。这些细胞中EPO需求消失的时间恰好与细胞增殖的显著下降相吻合。Northern分析表明，EPO受体信使RNA水平在第8天至第10天略有下降，第10天至第14天迅速下降。125I-EPO与红细胞的结合也显示在成熟和终末分化过程中细胞表面结合稳步下降。在转录抑制剂放线菌素D存在的情况下，人EPO受体的半衰期为90分钟，在8至14天的培养期间，在两个不同的时间测量的半衰期保持不变。这些结果表明，人EPO受体mRNA必须持续转录，才能保持Northern分析所看到的水平。这里描述的人类细胞系统非常适合于研究红系分化晚期的各种生化事件。
Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.