Fibach E
Department of Hematology, Hadassah University Hospital Ein-Kerem, Jerusalem, Israel.
Hemoglobin. 1998 Sep-Nov;22(5-6):445-58. doi: 10.3109/03630269809071542.
This report describes in detail the procedures for growing human erythroid cells in liquid culture for evaluating the potential of pharmacological agents to affect hemoglobin production. The procedure consists of two phases: an erythropoietin-independent phase in which peripheral blood mononuclear cells are first cultured in the presence of a combination of hemopoietic growth factors, but in the absence of erythropoietin, where early erythroid committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an erythropoietin-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. This procedure produces large cultures of relatively pure and synchronized erythroid cell populations derived from normal donors or patients with beta hemoglobinopathies. The cultured cells recapitulate many aspects of erythropoiesis in vivo, including the donor's pattern of hemoglobin production (types and proportions). Tested compounds, at different concentrations, are added at different stages of the culture. The various types of hemoglobins and globin chains produced can be measured by high performance liquid chromatographic techniques and their cellular distribution analyzed by flow cytometry using fluorescently labeled antibodies against specific hemoglobins. This approach provides a screening system for compounds with potential therapeutic efficacy in patients with beta hemoglobinopathies.
本报告详细描述了在液体培养中培养人红细胞系细胞的程序，以评估药理试剂影响血红蛋白产生的潜力。该程序包括两个阶段：一个不依赖促红细胞生成素的阶段，在此阶段外周血单个核细胞首先在造血生长因子组合存在但无促红细胞生成素的情况下进行培养，早期红细胞定向祖细胞在此增殖并分化为更成熟的祖细胞。在第二阶段，将后者细胞在添加促红细胞生成素的培养基中培养，继续增殖并成熟为正染性晚幼红细胞和无核红细胞。该程序可产生大量相对纯净且同步的红细胞系细胞群体培养物，这些细胞群体来源于正常供体或β地中海贫血患者。培养的细胞概括了体内红细胞生成的许多方面，包括供体的血红蛋白产生模式（类型和比例）。在培养的不同阶段添加不同浓度的受试化合物。产生的各种类型的血红蛋白和珠蛋白链可通过高效液相色谱技术进行测量，其细胞分布可通过使用针对特定血红蛋白的荧光标记抗体的流式细胞术进行分析。这种方法为β地中海贫血患者提供了一种对具有潜在治疗功效的化合物进行筛选的系统。
