Separation of functionally distinct human granulocyte-macrophage colony-stimulating factors.
Separation of functionally distinct human granulocyte-macrophage colony-stimulating factors.
复制标题
分离功能不同的人粒细胞-巨噬细胞集落刺激因子。
作者：
N. Nicola;D. Metcalf;G. Johnson;A. Burgess
人胎盘条件培养基（HPCM）含有人类骨髓嗜中性粒细胞-巨噬细胞（GM）和嗜酸性粒细胞（EO）祖细胞体外生长所需的集落刺激因子（csf）。通过在苯基- sepharose柱上操作洗脱条件，实现了HPCM中csf的分离。在高离子强度（1 M硫酸铵）下平衡苯基- sepharose柱后，所有CSF结合；一种GM-CSF （α）和所有可洗脱的EO-CSF仅通过降低盐浓度从柱中洗脱，而第二种GM-CSF （β）不含EO-CSF，仅通过增加洗脱缓冲液中乙二醇的浓度即可洗脱。两种gm - csf在功能上是不同的。培养第14天，GM-CSF α优先刺激菌落形成，随着刺激强度的降低，中性粒细胞菌落比例降低，巨噬细胞菌落比例增加；另一方面，GM-CSF β在培养第7天优先刺激菌落形成，中性粒细胞菌落比例高（平均80%），与GM-CSF β的浓度无关。根据tel过滤柱上的表观分子大小（分子量为30,000）和等电聚焦床上的电荷性质（等电点，4.9），GM-CSF α和GM-CSF β无法区分，并且彼此之间没有相关性，因为唾液糖蛋白与其亚洲糖蛋白形式相关。靶骨髓细胞的贴壁细胞去除（去除集落刺激细胞）表明，这两种GM-CSF直接起作用，而不是通过刺激GM-CSF的产生。混合和滴定实验表明，两种GM-CSF的功能特异性差异（以及与GM-CSF β相关的EO-CSF的缺乏）不是由于特定抑制分子的存在或一个部分相对于另一个部分的CSF的绝对水平较低。这两种GM-CSF可用于单独枚举不同的gm -祖细胞亚群。
Human placental conditioned medium (HPCM) contans colony-stimulating factors (CSFs) required for the growth in vitro of neutrophilic granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from human bone marrow. Fractionation of CSFs in HPCM was achieved by manipulation of the elution conditions on a column of phenyl-Sepharose. After equilibration of the phenyl-Sepharose column at high ionic strength (1 M ammonium sulfate), all of the CSF bound; one species of GM-CSF (alpha) and all of the elutable EO-CSF were eluted from the column simply by reducing the salt concentration, whereas the second species of GM-CSF (beta) was free of EO-CSF and was eluted only by increasing the concentration of tehylene glycol in the elution buffer. The two GM-CSFs were functionally distinct. GM-CSF alpha preferentially stimulated colony formation by day 14 of culture, and there was a decreased proportion of neutrophil colonies and increased proportion of macrophage colonies as the strength of the stimulus was decreased; GM-CSF beta, on the other hand, preferentially stimulated colony formation by day 7 of culture, and the proportion of neutrophil colonies was high (average 80%) and independent of the concentration of GM-CSF beta. GM-CSF alpha and GM-CSF beta were indistinguishable on the basis of apparent molecular size on tel filtration columns (molecular weight 30,000), charge properties on isoelectric focusing beds (isoelectric point, 4.9), and were not related to each other as a sialoglycoprotein is related to its asialo form. Adherent cell removal of the target bone marrow cells (to remove colony-stimulating cells) suggested that both GM-CSFs acted directly rather than by stimulating the production of GM-CSF. Mixing and titration experiments indicated that the differences in functional specificities of the two GM-CSFs (and the lack of EO-CSF associated with GM-CSF beta) were not due to the presence of specific inhibitory molecules or lower absolute levels of CSF in one fraction relative to the other. These two species of GM-CSF should be useful in separately enumerating subpopulations of different GM-progenitor cells inhuman hemopoietic disorders.