Studies on soluble granulocyte colony-stimulating factor (G-CSF) receptor

. 1。人血清与生物素化的G-CSF孵育后，样品进行天然聚丙烯酰胺凝胶电泳（SDS-PAGE）。电泳蛋白通过电印迹转移到硝化纤维素膜上，并与过氧化物酶（PO）标记的链亲和素反应。PO反应提示G-CSF结合物质的存在。检测到两个分子，一个为95kD，另一个为105kD。在减去G-CSF本身的20kD后，这些分子分别为75kd和85kd。此外，我们通过cDNA结构推导出G-CSF受体蛋白不同结构域的多肽抗体。b .可溶性G-CSF受体的结构用逆转录聚合酶链式反应（RT-PCR）研究了G-CSF受体在中性粒细胞不同成熟状态下的表达状态。由于可溶性G-CSF受体可能由II型G-CSF受体cDNA编码（通过选择性剪接形成），我们使用引物覆盖整个跨膜结构域，以不同大小的条带检测II型G-CSF受体cDNA（截断型）和I型G-CSF受体cDNA（野生型，全长）。用流式细胞仪将骨髓中性粒细胞系细胞分离到4个成熟阶段细胞群，发现成熟阶段小分子量带（可溶性G-CSF受体）的强度增加。c .可溶性G-CSF受体的作用用转染cDNA的大肠杆菌制备的可溶性小鼠G-CSF受体的作用检测。将其添加到体外培养的人骨髓中，粒细胞集落的形成明显受到抑制。这种溶解的小鼠G-CSF也能抑制人髓系白血病细胞的增殖。此外，我们还发现了一种新型可溶性G-CSF受体，来自严重的先天性中性粒细胞减少症患者。这种新的变异也在人髓性白血病细胞系中检测到。虽然急性髓母细胞白血病患者经常观察到严重的正常粒细胞生成抑制，但可溶性G-CSF受体的变异形式可能对正常粒细胞生成而白血病细胞具有很强的增殖抑制活性。为了证明这一假设，可溶性G-CSF受体cDNA的新变体形式已在哺乳动物CHO细胞中表达。少

A.1.Detection of soluble G-CSF receptorAfter incubation of human serum with biotinylated G-CSF,samples were subjected to native rplyacrylamide gel electrophorcsis (SDS-PAGE). Electrophoresed proteins were transferred to nitrocllulose membrane by electroblotting and peroxidase (PO) labeled streptavidin was reacted. PO reaction reveled the existence of G-CSF binding substances. Two molecules were detected, one was 95kD and the other was 105kD.After subtracting 20kD of G-CSF itself, these molecules were 75kd and 85kd, respectively. Furthermore, we developed antibodies against the polypeptides of various domains of G-CSF receptor protein deduced by cDNA structure. Similar results were obtained using these antitibodies and these findings indicate the existence of soluble G-CSF receptor at physiological condition.B.Structure of soluble G-CSF receptorExpression states of G-CSF receptor on various maturation states of neutrophilic graunlocytes were studies with reverse transcription polymerase … More chain reactions (RT-PCR). Since soluble G-CSF receptor was probably coded by type II G-CSF receptor cDNA (formed by alternative splicing), we used the primers to encompass whole transmembrane domain to detect type II G-CSF receptor cDNA (truncated type) as well as type I (wild type, full length) G-CSF receptor cDNA as different size bands. We fractionated bone marrow neutrophil lineage cells with flowcytometric cell sorter to 4 maturation stage cell populations and found the intensity of small molecular weight band (soluble G-CSF receptor) increased during maturation stages.C.Action of soluble G-CSF receptorAction of soluble murine G-CSF receptor which was made from the cDNA transfected E.coli was examined. When it was added to human bone marrow cultue in vitro, the formation of granulocyte colony was significantly suppressed. Human myeloid leukemic cell proliferation was also suppressed by this solublized murine G-CSF.Furthermore, we found new type soluble G-CSF receptor from a patient with severe congenital neutropenia. This new variant also detected in human myeloid leukemia cell lines. While severe suppression of normal granulopoiesis is often observed in patients with acute myeloblastic leukemia, the variant form of soluble G-CSF receptor may possess strong proliferation suppressing activity to normal granulopoiesis but leukemic cells. To prove this hypothesis, the new variant form of soluble G-CSF receptor cDNA has been expressed in mammalian CHO cells. Less