Ryu K H, Chun S, Carbonierre S, Im S A, Kim H L, Shin M H, Shin H Y, Ahn H S, Woo S Y, Seoh J Y, Fraser J K
Department of Paediatrics, Medical Research Centre, College of Medicine, Ewha Womans University, Seoul, Korea.
Br J Haematol. 2001 May;113(2):470-8. doi: 10.1046/j.1365-2141.2001.02762.x.
Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.
血小板生成素（TPO）是巨核细胞生成的主要调节因子，在早期造血过程中发挥着重要作用。此前，我们已经证明TPO在脐血（CB）CD34+细胞的体外扩增过程中诱导了一种特征性的凋亡模式。在本研究中，我们证明TPO诱导的凋亡细胞属于巨核细胞（MK）谱系，并且最初扩增的MK祖细胞随着TPO诱导凋亡的出现而减少。人CB CD34+细胞在含TPO的无血清条件下进行扩增。通过同时测量凋亡和免疫表型分析的多维流式细胞术表明，TPO诱导的凋亡细胞出现在CD61+组分中。对荧光激活细胞分选组分的免疫细胞化学分析表明，与凋亡相关的CD44low组分表达CD61。集落形成试验显示，在最初的9天内，巨核细胞集落形成单位（CFU-MK）总数增加了7.4±0.50倍。此后，CFU-MK的数量随着凋亡的增加而平行减少。当根据大小对MK集落进行细分时，大集落的比例逐渐降低，而中集落和小集落的比例增加。特别是从第6天起，小集落占主导地位。这些结果表明，MK祖细胞在与TPO进行体外扩增时随着扩增而成熟，然后进入凋亡阶段。
