Dolzhanskiy A, Basch R S, Karpatkin S
Department of Pathology, New York University Medical Center, NY 10016, USA.
Blood. 1997 Jan 15;89(2):426-34.
Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human bone marrow with a purity greater than 98% and a viability of 95%, using affinity techniques with magnetic beads followed by fluorescence-activated cell sorting. These cells were incubated in synthetic media containing the cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor (SCF), and IL-6, obviating the confounding effects of serum growth factors or cytokine secretions of non-MK cells on MK maturation. MK number, MK colony-forming units (CFU-MK), and MK ploidy and phenotype were examined during 7 days in culture. TPO in serum-free cultures without any other exogenously added cytokine supported MK growth and maturation. SCF synergized with TPO to augment MK production and maturation and could partially replace it under some conditions. Both TPO and IL-3 alone increased MK number (12- and 5-fold, respectively) and CFU-MK (approximately 15-fold each). SCF alone had no effect on MK proliferation in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to 2.0-fold in the presence of TPO. When combined with IL-3, SCF increased both MK number and CFU-MK by 15- to 20-fold in the absence of TPO. In the presence of TPO, the combination of IL-3 and SCF produced only modest increases (1.5- to 2.0-fold) in both MK number and CFU-MK. The proportion of polyploid MK increased greater than fivefold in the presence of TPO. SCF had little effect on MK ploidy in the presence of TPO, but enhanced ploidy twofold to threefold in the absence of TPO. IL-3 alone never increased the level of polyploidization. Rather, it consistently inhibited TPO- and SCF-induced polyploidization of MK. This inhibition was observed in cultures with or without SCF or IL-6. Although IL-3 also supported the proliferation of CD41+ cells and CFU-MK production, the cells that developed under the influence of IL-3 were phenotypically unusual (CD41dim, CD42dim) and of relatively low ploidy. Mature MK were not produced. When added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates MK growth and maturation, whereas IL-3 stimulates growth without maturation and may serve to conserve the immature MK compartment.
使用磁珠亲和技术结合荧光激活细胞分选，从人骨髓中分离出巨核细胞（MK）祖细胞，即CD34+CD41+细胞，其纯度大于98%，活力为95%。将这些细胞在含有细胞因子血小板生成素（TPO）、白细胞介素-3（IL-3）、干细胞因子（SCF）和IL-6的合成培养基中培养，避免了血清生长因子或非MK细胞的细胞因子分泌对MK成熟的混杂影响。在培养的7天内检测MK数量、MK集落形成单位（CFU-MK）以及MK的倍性和表型。在无任何其他外源性添加细胞因子的无血清培养中，TPO支持MK的生长和成熟。SCF与TPO协同作用以增加MK的产生和成熟，并且在某些条件下可以部分替代TPO。单独的TPO和IL-3均可增加MK数量（分别增加12倍和5倍）和CFU-MK（各自约增加15倍）。在无TPO时，单独的SCF对MK增殖无影响，但在有TPO时可使MK数量和CFU-MK增加1.5至2.0倍。与IL-3联合时，在无TPO的情况下，SCF可使MK数量和CFU-MK增加15至20倍。在有TPO时，IL-3和SCF的联合仅使MK数量和CFU-MK有适度增加（1.5至2.0倍）。在有TPO时，多倍体MK的比例增加超过五倍。在有TPO时，SCF对MK倍性影响较小，但在无TPO时可使倍性增加两倍至三倍。单独的IL-3从未增加多倍体化水平。相反，它始终抑制TPO和SCF诱导的MK多倍体化。在有或无SCF或IL-6的培养中均观察到这种抑制作用。尽管IL-3也支持CD41+细胞的增殖和CFU-MK的产生，但在IL-3影响下发育的细胞在表型上不寻常（CD41dim，CD42dim）且倍性相对较低，未产生成熟的MK。当与TPO一起添加时，IL-3抑制多倍体化。因此，TPO刺激MK生长和成熟，而IL-3刺激生长但不促进成熟，可能有助于维持未成熟的MK区室。
