血小板因子-4抑制U937细胞增殖的机理研究

发布时间：2018-05-11 20:49

  本文选题：血小板因子4 + CXC受体3-B　； 参考：《中国人民解放军军事医学科学院》2005年博士论文

【摘要】：血小板因子4(Platelet factor 4,PF4)是作用最强的抑制血管增生的趋化因子,但其作用机理一直不明,可能与以下三种机理相关:1.通过与蛋白多糖的结合来干扰蛋白多糖对生长因子活性的作用;2.直接与血管增生刺激因子结合,以抑制它们与相应受体的作用;3.通过活化或结合细胞表面的受体,启动相应的负调节信号转导通路。本研究首次发现PF4可以在体外直接抑制人淋巴瘤细胞系U937的生长,但对人白血病细胞系K562、HL-60却无明显影响。构建PF4突变体(PTA37/38/39AVP、R49S、L55R、A57V)并检测它们对U937细胞的增殖影响,结果显示除A57V之外,其余3个突变体均失去抑制U937细胞增殖的活性,这3个突变体除R49S与肝素结合相关外,其余2个均与肝素结合无关,说明PF4抑制U937细胞生长与第37、38、39、49、55位的氨基酸密切相关,且这种抑制作用可能是非肝素依赖性的。这提示PF4抑制U937细胞增殖可能与第三种机理相关。2003年,Lasagni等首次报道了PF4的唯一受体CXCR3—B,PF4与CXCR3—B结合,可抑制内皮细胞的增殖,为研究PF4抑制肿瘤生长的机理提供了新的思路。本研究旨在以CXCR3—B为线索,探讨PF4抑制肿瘤细胞增殖的可能的作用机理。首先制备了兔/鼠抗人CXCR3—B分子的多/单克隆抗体,并通过三个不同区段的多肽对单克隆抗体的抗原表位进行了初步的筛选。共获得三种针对不同区段的多肽的多/单克隆抗体,这些抗体为进行有关PF4抑制U937细胞增殖的作用机理研究奠定了基础。第二,通过RT—PCR、免疫印迹及免疫沉淀的方法证实U937细胞表达CXCR3—B分子,Pull-down、流式细胞术的结果证明PF4可识别CXCR3—B分子并与之结合,说明PF4抑制U937细胞的增殖可能是通过CXCR3—B介导的。第三,通过蛋白组学和基因芯片的实验技术对PF4作用于U937细胞后的蛋白及表达变化基因进行了初步的筛选。结果显示PF4可从U937细胞总蛋白中富集分子量约为75kDa的蛋白,此蛋白可能是ZNF224或是前髓白血病锌指蛋白,
[Abstract]:Platelet factor 4(Platelet factor 4 (PF4) is the most potent chemokine to inhibit angiogenesis, but its mechanism is still unknown, which may be related to the following three mechanisms: 1: 1. It interferes with the effect of proteoglycan on growth factor activity by binding with proteoglycan. It binds directly to angiogenesis stimulating factors to inhibit their effects on the corresponding receptors. By activating or binding the receptors on the cell surface, the corresponding negative regulated signal transduction pathway is initiated. In this study, we first found that PF4 could directly inhibit the growth of human lymphoma cell line U937 in vitro, but had no effect on human leukemia cell line K562 HL-60. Construction of the PF4 mutant PTA37 / 38 / 39 AVPN R49SN L55RH5 A57V) and their effects on the proliferation of U937 cells were examined. The results showed that the other three mutants except A57V lost the activity of inhibiting the proliferation of U937 cells. The three mutants except R49S were associated with heparin. The other two were not associated with heparin binding, suggesting that the inhibition of U937 cell growth by PF4 was closely related to the amino acid at position 37,38-39, 49.55.The inhibitory effect may be heparin-independent. It is suggested that the inhibition of U937 cell proliferation by PF4 may be related to the third mechanism. In 2003, Lasagni et al reported for the first time that CXCR3-BNPF4, the only receptor of PF4, binds to CXCR3-B, which can inhibit the proliferation of endothelial cells. It provides a new way to study the mechanism of PF4 inhibiting tumor growth. The aim of this study was to explore the possible mechanism of PF4 inhibiting tumor cell proliferation based on CXCR3-B. Firstly, rabbit / mouse polyclonal antibodies against human CXCR3-B molecules were prepared, and the epitopes of monoclonal antibodies were preliminarily screened by three different polypeptides. Three polyclonal antibodies against different polypeptides were obtained, which laid a foundation for the study of the mechanism of PF4 inhibiting the proliferation of U937 cells. Secondly, U937 cells expressed CXCR3-B molecules Pull-downvia RT-PCR, Western blot and immunoprecipitation. Flow cytometry showed that PF4 could recognize and bind CXCR3-B molecules. The results suggest that PF4 inhibits the proliferation of U937 cells by CXCR3-B. Thirdly, the protein and expression change genes of U937 cells treated with PF4 were preliminarily screened by proteomics and gene chip techniques. The results showed that PF4 could enrich the protein with molecular weight of 75kDa from the total protein of U937 cells. This protein may be ZNF224 or promyelocytic zinc finger protein.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392
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本文编号：1875542