Heinrich M C, Dooley D C, Freed A C, Band L, Hoatlin M E, Keeble W W, Peters S T, Silvey K V, Ey F S, Kabat D
Department of Medicine, Oregon Health Sciences University, Portland 97201-3098.
Blood. 1993 Aug 1;82(3):771-83.
Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.
钢因子（SF）是c-kit的配体，是正常造血、黑色素生成、配子发生以及肥大细胞生长和发育的重要调节因子。造血基质细胞是SF的重要来源，因为小鼠中SF的失活会导致造血基质细胞支持功能的缺陷。为了确定在人类造血过程中产生SF的特定细胞以及控制SF不同异构体表达的因子，我们在人基质细胞暴露于重组人白细胞介素（IL）-1α（一种已知可诱导多种造血生长因子表达的细胞因子）之前和之后，对SF mRNA和膜结合蛋白水平进行了定量。此外，由于长期骨髓培养（LTBMC）中的基质细胞在体外支持造血祖细胞存活，而脐静脉内皮细胞（EC）和二倍体成纤维细胞（DF）则不支持，我们还试图检验这样一个假设，即与EC或DF相比，LTBMC细胞中的SF基因表达会有所不同。使用逆转录聚合酶链反应扩增（RT-PCR）、核糖核酸酶保护分析（RPA）和Northern印迹分析，发现SF在EC、DF和LTBMC中组成性转录。IL-1α既未诱导这些基质细胞中SF mRNA的积累，也未改变外显子6 +与外显子6 -转录本的比例。通过Northern印迹分析，显示主要的SF mRNA种类为5.6 kb；还发现了一小部分3.6 kb的。发现所有三种类型的基质细胞都组成性表达低水平的膜结合SF蛋白，并且不受IL-1α的调节。我们得出结论，与EC或DF相比，LTBMC在体外支持造血的独特能力不能基于这些细胞中SF基因表达的定性或定量差异来解释。
