Stem cell growth factor: isolation of the cDNA, analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombmant preparation on primitive hematopoietic progenitor cells

为了实现干细胞生长因子(SCGF)在血液学中的诊断和治疗应用，我们研究了一种重组人(Rh)SCGF的分子特征、基因表达和造血活性。分别以骨髓细胞、MC3T3-G2/PA6前脂肪细胞和ROS-17/2.8-5骨肉瘤细胞为材料，克隆了人、小鼠和大鼠SCGF的全长cDNA。SCGF是C型凝集素超家族的新成员，与四环素同源。去掉一个21aa信号肽，得到相对分子质量为40 kDa的成熟SCGF。SCGF不是N-糖基化的，而是与唾液酸O-糖基化的。亮氨酸拉链状序列可诱导干细胞生长因子形成三元α-螺旋卷曲结构。Northern和原位杂交分析表明，SCGF mRNA在脾、胎肝和骨骼组织中特异表达，特别是在骨髓细胞、骨膜和增殖的软骨细胞中表达，而在静止或肥大的软骨细胞…中几乎没有表达更多的S，表明干细胞生长因子影响成骨。荧光原位杂交将人和小鼠的SCGF基因分别定位在染色体19q13.3和7B3-B5上，其中Flt3配体和IL-11基因聚在一起。用表达载体PAGE-SCGF稳定转化CHO细胞，并从培养上清液中纯化出无内毒素的重组人SCGF。用人工合成的寡肽免疫大鼠，获得抗SCGF的单抗。对于人骨髓CD34+LIN-细胞，在促红细胞生成素、GM-CSF或G-CSF的作用下，rhSCGF分别促进红系集落和粒/巨噬细胞集落的生长，并与IL-3和Flt3配体协同促进GM集落生长。为了进一步研究SCGF在其他细胞、受体、基因敲除小鼠和临床方面的作用，SCGF有望建立一种新的干细胞生物学范式。较少

On an attempt to realize diagnostic and therapeutic application of stem cell growth factor (SCGF) in hematology we studied molecular characterization, the gene expression and hematopoietic activities of a recombmant human (rh) SCGF. A full-length cDNA for human, mouse and rat SCGF was cloned using bone marrow cells, MC3T3-G2/PA6 preadipocytes and ROS-17/2.8-5 osteosarcoma cells, respectively, SCGF was a new member of a C-type lectin superfamily and most homologous with tetranectin. A 21aa signal peptide was removed to give rise to mature SCGF with MW 40 kDa. SCGF was not N-glycosylated, but O-glycosylated with sialic acid. Leucine zipper-like sequence could induce SCGF to form a ternary α-helical coiled-coil structure. Northern and in situ hybridization analysis showed specific expression of SCGF mRNA in the spleen, fetal liver and skeletal tissues, particularly in bone marrow cells, periosteum and proliferating chondrocytes, but little expression in resting or hypertrophic chondrocyte … More s, indicating that SCGF affected osteogenesis. Fluorescent in situ hybridization mapped human and mouse SCGF genes to chromosome 19q13.3 and 7B3-B5, respectively, where flt3 ligand and IL-11 genes were clustered. CHO cells were stably transformed with an expression vector PAGE-SCGF, and an endotoxin-free rhSCGF was purified from the culture supernatant. Anti-SCGF monoclonal antibodies were obtained by immunizing rats with a synthetic oligopeptide. For human bone marrow CD34+Lin- cells rhSCGF promoted growth of erythroid and granulocyte/macrophage colonies in the culture with erythropoietin and GM-CSF or G-CSF, respectively, and was synergistic with IL-3 and flt3 ligand to enhance GM colony-growth.SCGF is selectively produced by osseous and hematopoietic stromal cells and can mediate their proliferative activity on hematopoietic stem cells. For further characterization of SCGF, activities on other cells, receptor, knockout mice and clinical aspects should be studied, and SCGF is surely expected to build a new stem cell biology paradigm. Less