Kohama T, Handa H, Harigaya K
Department of Pathology, Keio University School of Medicine, Tokyo, Japan.
Exp Hematol. 1988 Aug;16(7):603-8.
Recently, several human bone marrow stromal cell lines have established and produced hematopoietic growth factors. One of these factors, a burst-promoting activity (BPA), was purified from 6 liters of serum-free conditioned medium cultured from stromal cell line KM-102, which was stimulated by phorbol myristate acetate (PMA) and calcium ionophore A23187. This stimulation induced 60 times more production of BPA than the unstimulated control culture. BPA was purified 4000-fold by sequential fractionation using ammonium sulfate precipitation, anion-exchange and lentil lectin affinity chromatographies, high performance gel filtration chromatography, and reversed phase high performance liquid chromatography. Purified BPA gave a single broad band of protein with a molecular weight of approximately 18 kd, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentration required for half maximal growth of early erythroid colonies was estimated as 10 pg/ml or 0.6 pM. At a higher concentration (125 pg/ml) this factor also stimulates the growth of granulocyte, macrophage, and eosinophil colonies in agar culture. The profile of amino acid composition is very similar to that of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) deduced from its complementary DNA sequence. The result of amino-terminal sequence analysis strongly suggests that the purified material consists of GM-CSF and tetrapeptide-deleted GM-CSF. Moreover, antibody against GM-CSF completely neutralized the biological activities of this factor. These results indicate that the human bone marrow stromal cell line secretes GM-CSF as a burst-promoting activity and GM-CSF may play a significant role in the interaction between stem cells and stromal cells in the hematopoietic microenvironment.
最近，已建立了几种人骨髓基质细胞系并产生造血生长因子。其中一种因子，即爆式促进活性（BPA），是从6升由基质细胞系KM - 102培养的无血清条件培养基中纯化得到的，该基质细胞系用佛波酯肉豆蔻酸酯（PMA）和钙离子载体A23187刺激。这种刺激诱导产生的BPA比未刺激的对照培养物多60倍。通过硫酸铵沉淀、阴离子交换和扁豆凝集素亲和层析、高效凝胶过滤层析以及反相高效液相层析的顺序分级分离，BPA被纯化了4000倍。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳评估，纯化的BPA呈现出一条单一的宽带状蛋白质，分子量约为18 kd。早期红系集落半数最大生长所需的浓度估计为10 pg/ml或0.6 pM。在较高浓度（125 pg/ml）时，该因子还能刺激琼脂培养中的粒细胞、巨噬细胞和嗜酸性粒细胞集落的生长。氨基酸组成谱与从其互补DNA序列推导的人粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）非常相似。氨基末端序列分析结果强烈表明，纯化的物质由GM - CSF和缺失四肽的GM - CSF组成。此外，抗GM - CSF抗体完全中和了该因子的生物学活性。这些结果表明，人骨髓基质细胞系分泌GM - CSF作为爆式促进活性，并且GM - CSF可能在造血微环境中干细胞与基质细胞之间的相互作用中发挥重要作用。
