Miyazaki H, Inoue H, Yanagida M, Horie K, Mikayama T, Ohashi H, Nishikawa M, Suzuki T, Sudo T
Pharmaceutical Research Laboratory, Kirin Brewery Company, Limited, Gunma, Japan.
Exp Hematol. 1992 Aug;20(7):855-61.
We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.
我们最近报道了四种抗大鼠血小板糖蛋白IIb/IIIa（GPIIb/IIIa）单克隆抗体（MoAb）的制备及特性。在本研究中，我们基于使用一种MoAb即P55通过免疫吸附（淘选）进行阳性选择，开发了一种简单高效的三步程序，以从正常骨髓中纯化大鼠巨核细胞集落形成细胞（巨核细胞集落形成单位，CFU-MK）。在软琼脂培养中，于伴刀豆球蛋白A（Con A）刺激的大鼠脾细胞条件培养基存在下，对每一步获得的细胞形成巨核细胞集落的能力进行检测。骨髓细胞首先在不连续的Percoll梯度上进行分离。密度在1.063至1.082 g/ml之间沉淀的细胞去除了粘附于塑料组织培养皿的细胞。非粘附细胞进一步在包被有P55 MoAb的培养皿上孵育。CFU-MK在吸附细胞部分中富集了约50倍。这种顺序分级分离程序使大鼠CFU-MK相对于全骨髓细胞富集了345倍（范围为276至412倍）。最终部分中CFU-MK的平均克隆效率约为有核细胞的7%（范围为5% - 9.2%）。CFU-MK的总体回收率平均为20%（范围为9% - 29%）。淘选步骤使巨核细胞爆式集落形成细胞（巨核细胞爆式集落形成单位，BFU-MK）富集了46倍，其在淘选后部分中的平均克隆效率为0.14%（范围为0.07% - 0.2%）。此外，红系爆式集落形成细胞（红系爆式集落形成单位，BFU-E）也通过淘选得到显著富集，但程度低于BFU-MK和CFU-MK。相比之下，粒细胞 - 巨噬细胞集落形成细胞（粒细胞 - 巨噬细胞集落形成单位，CFU-GM）和红系集落形成细胞（红系集落形成单位，CFU-E）未通过淘选得到富集。淘选后获得的CFU-MK在重组大鼠白细胞介素3（rIL-3）、小鼠粒细胞 - 巨噬细胞集落刺激因子（mGM-CSF）或人促红细胞生成素（hEPO）存在下形成巨核细胞集落，正如全骨髓细胞中的鼠CFU-MK所报道的那样。因此，高度富集的大鼠CFU-MK群体应为进一步研究巨核细胞生成的调控提供基础。
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