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

A. 1.可溶性G-CSF受体的检测将人血清与生物素化的G-CSF孵育后，对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酸纤维素膜上，过氧化物酶（PO）标记的链霉亲和素反应。PO反应显示G-CSF结合物的存在。结果表明，G-CSF的分子量分别为95 kD和105 kD，扣除G-CSF本身的20 kD后，分子量分别为75 kD和85 kD。此外，我们开发了针对由cDNA结构推导的G-CSF受体蛋白的各个结构域的多肽的抗体。B.可溶性G-CSF受体的结构用逆转录聚合酶研究了G-CSF受体在不同成熟状态的嗜中性粒细胞上的表达状态 关于我们 链反应（RT-PCR）。由于可溶性G-CSF受体可能是由II型G-CSF受体cDNA（通过选择性剪接形成）编码的，我们使用包含整个跨膜结构域的引物来检测II型G-CSF受体cDNA（截短型）以及I型（野生型，全长）G-CSF受体cDNA作为不同大小的条带。我们用流式细胞仪将骨髓中性粒细胞系细胞分成4个成熟期细胞群，发现在成熟期细胞中小分子量条带（可溶性G-CSF受体）的强度增加。C.可溶性G-CSF受体的作用检测了由cDNA转染的大肠杆菌制备的可溶性鼠G-CSF受体的作用。将其加入体外培养的人骨髓中，可明显抑制粒细胞集落的形成。此外，我们还从一例严重先天性中性粒细胞减少症患者体内发现了一种新型的可溶性G-CSF受体。这种新的变异也在人类骨髓性白血病细胞系中检测到。虽然在急性髓性白血病患者中经常观察到正常粒细胞生成的严重抑制，但可溶性G-CSF受体的变体形式可能对正常粒细胞生成而不是白血病细胞具有强的增殖抑制活性。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