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

A.1.可溶性G-CSF受体的检测人血清与生物素标记的G-CSF孵育后，样品进行天然聚丙烯酰胺凝胶电泳(SDS-PAGE)。用电印迹法将电泳后的蛋白质转移到硝基纤维膜上，再用过氧化物酶(PO)标记链霉亲和素。Po反应证实了G-CSF结合物质的存在。检测到两个分子，一个是95kD，一个是105kD，减去G-CSF本身的20kD后，这两个分子分别为75kD和85kD。此外，我们还研制了针对G-CSF受体蛋白不同结构域多肽的抗体。B.用逆转录聚合酶链式反应(RT-…)研究中性粒细胞不同成熟状态下G-CSF受体的表达状态更多的连锁反应(RT-PCR)。由于可溶性粒细胞集落刺激因子受体可能是由II型G-CSF受体基因编码的(选择性剪接形成)，所以我们用该引物包被整个跨膜区，以不同大小的条带来检测II型G-CSF受体基因(截短型)和I型(野生型全长)G-CSF受体基因。我们用流式细胞仪将骨髓中性粒细胞分为4个成熟期细胞群，发现小分子量带(可溶性G-CSF受体)的强度在成熟期增加。C.可溶性G-CSF受体的作用检测从转基因大肠杆菌中制备的可溶性小鼠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形成。该可溶性G-CSF也能抑制人髓系白血病细胞的增殖。此外，我们还从一例严重的先天性中性粒细胞减少症患者身上发现了新型的可溶性G-CSF受体。这种新的变种也在人类髓系白血病细胞系中检测到。急性髓系白血病患者常表现为正常粒系严重抑制，但其变异型G-CSF受体可能对正常粒系细胞有较强的增殖抑制活性，但对白血病细胞无明显抑制作用。为了证明这一假说，我们在哺乳动物CHO细胞中表达了新的可溶型G-CSF受体基因。较少

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