Kohsaki M, Noguchi K, Araki K, Horikoshi A, Sloman J C, Miyake T, Murphy M J
Proc Natl Acad Sci U S A. 1983 Jun;80(12):3802-6. doi: 10.1073/pnas.80.12.3802.
Significant in vivo stimulation of granulopoiesis was induced in mice by the administration of an extract from the urine of patients with aplastic anemia (AA). Sialic acid has been identified as an important molecular component for the in vivo biological activity of this granulopoietic factor, "granulopoietin," which is distinct and different from endotoxin. Urine from patients with AA was successively fractionated by Sephadex G-50 and DEAE-cellulose chromatography. The resultant extract, which we refer to as AA urinary extract, contained approximately equal to 44 international units of erythropoietin per A unit of protein and induced 15,000 colonies of granulocyte/macrophage precursor cells (granulocyte/macrophage colony-forming units, CFU-gm) per A unit of protein with mouse bone marrow. Eight daily intraperitoneal injections of this extract in mice induced a 6.2-fold increase in peripheral blood granulocytes and a 14.6-fold increase of splenic CFU-gm, with concomitant increases in the proliferation rates of CFU-gm in both bone marrow and spleen. Pretreatment of the AA urinary extract with sialidase significantly diminished these granulopoietic effects in vivo (P less than 0.001). In contrast, both extracts (i.e., native AA and sialidase-treated AA urinary extracts) revealed high granulocyte/macrophage colony-stimulating factor activity in vitro when clonal assays were performed with mouse bone marrow. Increased in vivo and in vitro granulopoietic activities were found in the concanavalin A "break-through" fraction, indicating that these activities were due to protein(s) that did not bind to the lectin. These results reveal that this urinary extract from patients with AA is capable of inducing significant granulopoiesis in mice and that sialic acid is an important component in the maintenance of this granulopoietic effect in vivo but not in vitro.
通过给予再生障碍性贫血（AA）患者尿液提取物，可在小鼠体内诱导显著的粒细胞生成刺激。唾液酸已被确定为这种粒细胞生成因子“粒细胞生成素”体内生物活性的重要分子成分，它与内毒素不同。AA患者的尿液先后通过葡聚糖G - 50和二乙氨基乙基纤维素色谱进行分级分离。所得提取物，我们称之为AA尿提取物，每A单位蛋白质中约含有44国际单位的促红细胞生成素，并且每A单位蛋白质能诱导小鼠骨髓中15,000个粒细胞/巨噬细胞前体细胞集落（粒细胞/巨噬细胞集落形成单位，CFU - gm）。每天对小鼠进行8次腹腔注射该提取物，可使外周血粒细胞增加6.2倍，脾CFU - gm增加14.6倍，同时骨髓和脾脏中CFU - gm的增殖率也随之增加。用唾液酸酶预处理AA尿提取物可显著降低其在体内的这些粒细胞生成作用（P小于0.001）。相比之下，当用小鼠骨髓进行克隆分析时，两种提取物（即天然AA尿提取物和经唾液酸酶处理的AA尿提取物）在体外均显示出高粒细胞/巨噬细胞集落刺激因子活性。在伴刀豆球蛋白A“突破”组分中发现体内和体外粒细胞生成活性均增加，这表明这些活性是由不与凝集素结合的蛋白质引起的。这些结果表明，AA患者的这种尿提取物能够在小鼠体内诱导显著的粒细胞生成，并且唾液酸是维持这种粒细胞生成作用在体内而非体外的重要成分。
