本文選題：血小板因子4 + CXC受體3-B　； 參考：《中國人民解放軍軍事醫(yī)學科學院》2005年博士論文

【摘要】：血小板因子4(Platelet factor 4,PF4)是作用最強的抑制血管增生的趨化因子,但其作用機理一直不明,可能與以下三種機理相關:1.通過與蛋白多糖的結合來干擾蛋白多糖對生長因子活性的作用;2.直接與血管增生刺激因子結合,以抑制它們與相應受體的作用;3.通過活化或結合細胞表面的受體,啟動相應的負調節(jié)信號轉導通路。本研究首次發(fā)現(xiàn)PF4可以在體外直接抑制人淋巴瘤細胞系U937的生長,但對人白血病細胞系K562、HL-60卻無明顯影響。構建PF4突變體(PTA37/38/39AVP、R49S、L55R、A57V)并檢測它們對U937細胞的增殖影響,結果顯示除A57V之外,其余3個突變體均失去抑制U937細胞增殖的活性,這3個突變體除R49S與肝素結合相關外,其余2個均與肝素結合無關,說明PF4抑制U937細胞生長與第37、38、39、49、55位的氨基酸密切相關,且這種抑制作用可能是非肝素依賴性的。這提示PF4抑制U937細胞增殖可能與第三種機理相關。2003年,Lasagni等首次報道了PF4的唯一受體CXCR3—B,PF4與CXCR3—B結合,可抑制內(nèi)皮細胞的增殖,為研究PF4抑制腫瘤生長的機理提供了新的思路。本研究旨在以CXCR3—B為線索,探討PF4抑制腫瘤細胞增殖的可能的作用機理。首先制備了兔/鼠抗人CXCR3—B分子的多/單克隆抗體,并通過三個不同區(qū)段的多肽對單克隆抗體的抗原表位進行了初步的篩選。共獲得三種針對不同區(qū)段的多肽的多/單克隆抗體,這些抗體為進行有關PF4抑制U937細胞增殖的作用機理研究奠定了基礎。第二,通過RT—PCR、免疫印跡及免疫沉淀的方法證實U937細胞表達CXCR3—B分子,Pull-down、流式細胞術的結果證明PF4可識別CXCR3—B分子并與之結合,說明PF4抑制U937細胞的增殖可能是通過CXCR3—B介導的。第三,通過蛋白組學和基因芯片的實驗技術對PF4作用于U937細胞后的蛋白及表達變化基因進行了初步的篩選。結果顯示PF4可從U937細胞總蛋白中富集分子量約為75kDa的蛋白,此蛋白可能是ZNF224或是前髓白血病鋅指蛋白,
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392
，

本文編號：1875542