Lu L, Briddell R A, Graham C D, Brandt J E, Bruno E, Hoffman R
Department of Medicine, Indiana Elks Cancer Research Center, Indiana University School of Medicine, Indianapolis 46223.
Br J Haematol. 1988 Oct;70(2):149-56. doi: 10.1111/j.1365-2141.1988.tb02456.x.
Nonadherent low density T-lymphocyte depleted (NALT-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18-fold increase in CE over NALT- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin MK colony formation by My10 DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8/5 x 10(3) My10 DR+ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10 DR+ cells are largely devoid of these accessory cells.
使用得克萨斯红标记的My10和藻红蛋白偶联的抗HLA - DR单克隆抗体，在库尔特Epics 753染料激光系统上对来自正常供体的非贴壁低密度T淋巴细胞耗竭（NALT -）骨髓细胞进行分选，以获得富集的巨核细胞集落形成单位（CFU - MK）群体。对于同时表达高密度My10和低密度HLA - DR（My10 DR +）的细胞，CFU - MK克隆效率（CE）为1.1 +/- 0.5%。该程序使CE相较于NALT - 细胞提高了18倍。利用血清耗尽检测系统，测定了纯化的或重组的人造血生长因子，包括促红细胞生成素（Epo）、血小板生成刺激因子（TSF）、白细胞介素1α（IL - 1α）、粒细胞集落刺激因子（G - CSF）、粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）、巨噬细胞集落刺激因子（M - CSF或CSF - 1）和白细胞介素对My10 DR + 细胞形成MK集落的影响。单独的Epo、TSF、CSF - 1、IL - 1α或G - CSF均未使MK集落形成增加至基线以上（接种的5×10³个My10 DR + 细胞中，基线为2.5 +/- 0.8个集落）。相反，添加GM - CSF和IL - 3均增加了CFU - MK集落形成以及集落大小，在添加200单位/毫升的IL - 3和25单位/毫升的GM - CSF后出现最大刺激。在最大浓度时，IL - 3促进巨核细胞集落形成的能力比GM - CSF更强。GM - CSF和IL - 3的刺激作用也是相加的，因为这两种因子组合的作用近似于单独存在每种因子时集落形成的总和。因此，CFU - MK似乎表达HPCA - 1和HLA - DR抗原。这些研究还表明，GM - CSF和IL - 3是体外巨核细胞生成的重要调节因子，并且由于My10 DR + 细胞基本上不含这些辅助细胞，这些生长因子的活性不依赖于大量巨噬细胞或T细胞的存在。
