Granulocyte colony-stimulating factor (GCSF) is a member of the hematopoietic growth factor family that acts primarily on neutrophils and neutrophilic precursors to promote cell proliferation and differentiation. Although multiple GCSF genes have been found in teleosts, knowledge of their functions during fish hematopoietic development is still limited. Here, we report for the first time the molecular and functional characterization of two goldfish GCSFs (gfGCSF-a and gfGCSF-b). The open reading frame (ORF) of the gfGCSF-a and gfGCSF-b cDNA transcript consisted respectively of 624 bp and 678 bp with its ORF encoding 207 and 225 amino acids (aa), with a 17 aa signal peptide for each gene and a conserved domain of the IL-6 superfamily. Treatment of goldfish head kidney leukocytes (HKLs) with LPS increased gfGCSF-a and gfGCSF-b mRNA expression levels, also exposure of HKLs to either heat-killed or live A. hydrophila, induced transcriptional upregulation of gfGCSF-a and gfGCSF-b levels. Recombinant gfGCSF-a and gfGCSF-b protein (rgGCSF-a and rgGCSF-b) induced a dose-dependent production of TNFα and IL-1β from goldfish neutrophils. In vitro experiments showed rgGCSF-a and rgGCSF-b differentially promoted the proliferation and differentiation of leukocytes in goldfish. Furthermore, treatment of HKLs with rgGCSF-a showed significant upregulation of mRNA levels of the hematopoietic transcription factor GATA2, Runx1, MafB, and cMyb, while gfGCSF-b induces not only all four transcriptional factors mentioned above but also CEBPα. Our results indicate that goldfish GCSF-a and GCSF-b are important regulators of neutrophil proliferation and differentiation, which could stimulate different stages and lineages of hematopoiesis.

粒细胞集落刺激因子（GCSF）是造血生长因子家族的成员，主要作用于中性粒细胞和中性粒细胞前体，促进细胞增殖和分化。尽管在硬骨鱼中发现了多个 GCSF 基因，但对其在鱼类造血发育过程中的功能的了解仍然有限。在这里，我们首次报告了两种金鱼 GCSF（gfGCSF-a 和 gfGCSF-b）的分子和功能表征。gfGCSF-a和gfGCSF-b cDNA转录本的开放阅读框（ORF）分别由624 bp和678 bp组成，其ORF编码207和225个氨基酸（aa），每个基因有17个aa信号肽和一个IL-6 超家族的保守结构域。用 LPS 处理金鱼头肾白细胞 (HKL) 会增加 gfGCSF-a 和 gfGCSF-b mRNA 表达水平，同时将 HKL 暴露于热灭活或活的嗜水放线菌中，也会诱导 gfGCSF-a 和 gfGCSF-b 水平的转录上调。重组 gfGCSF-a 和 gfGCSF-b 蛋白（rgGCSF-a 和 rgGCSF-b）诱导金鱼中性粒细胞剂量依赖性地产生 TNFα 和 IL-1β。体外实验表明，rgGCSF-a和rgGCSF-b对金鱼白细胞的增殖和分化有不同的促进作用。此外，用rgGCSF-a处理HKL显示造血转录因子GATA2、Runx1、MafB和cMyb的mRNA水平显着上调，而gfGCSF-b不仅诱导上述所有四种转录因子，还诱导CEBPα。我们的研究结果表明，金鱼GCSF-a和GCSF-b是中性粒细胞增殖和分化的重要调节因子，可以刺激造血的不同阶段和谱系。