Angchaisuksiri P, Carlson P L, Day E B, Dessypris E N
Department of Medicine, H. H. McGuire Department of Veterans Affairs Medical Center, Richmond, VA 23249.
Exp Hematol. 1994 Jul;22(7):546-50.
To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.
为了研究复制与核内复制之间的时间关系，以及成熟巨核细胞中血小板特异性蛋白的出现与核内复制之间的关系，将高度富集造血祖细胞的外周血单核细胞在液体培养物和血浆凝块中培养，培养条件为白细胞介素-3（IL-3）和干细胞因子（SCF），或植物血凝素（PHA）刺激的血液单核细胞条件培养基。在血浆凝块中，巨核细胞（MK）集落最早在第5天出现，并在第8天达到最大值，而每个集落中的细胞数量一直增加到第10天，这表明MK集落形成只有一波。在液体培养物中，第一个免疫可识别的巨核细胞在第5天出现，且仅表达糖蛋白IIb/IIIa和血小板反应蛋白，但所有其他血小板特异性蛋白标志物在24小时内出现。将液体培养基中的细胞重新接种到血浆凝块中显示，第6天糖蛋白IIb/IIIa阳性细胞中有92±8%能够复制。然而，它们的复制潜能随年龄下降，因此在第6天至第11天之间，具有复制能力的巨核细胞百分比的对数与其培养年龄之间呈线性相关。第10天后复制完全停止。在IL-3存在的情况下，多倍体巨核细胞在糖蛋白IIb/IIIa表达的同时出现，并且巨核细胞在倍性类别的分布直到第20天保持不变。在PHA-白细胞条件培养基（PHA-LCM）存在的情况下，巨核细胞的倍性在第6天后向更高类别转变，核内复制过程在第10天完成。在第10天至第20天之间未观察到倍性分布的变化。这些发现表明，在源自巨核细胞集落形成单位（CFU-MK）的巨核细胞群体中，核内复制可在发育早期发生，与复制同步进行，并在巨核细胞耗尽其复制潜能之前完成。
