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

【摘要】：血小板因子4(Platelet factor 4,PF4)是作用最強(qiáng)的抑制血管增生的趨化因子,但其作用機(jī)理一直不明,可能與以下三種機(jī)理相關(guān):1.通過(guò)與蛋白多糖的結(jié)合來(lái)干擾蛋白多糖對(duì)生長(zhǎng)因子活性的作用;2.直接與血管增生刺激因子結(jié)合,以抑制它們與相應(yīng)受體的作用;3.通過(guò)活化或結(jié)合細(xì)胞表面的受體,啟動(dòng)相應(yīng)的負(fù)調(diào)節(jié)信號(hào)轉(zhuǎn)導(dǎo)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(xì)胞系U937的生長(zhǎng),但對(duì)人白血病細(xì)胞系K562、HL-60卻無(wú)明顯影響。構(gòu)建PF4突變體(PTA37/38/39AVP、R49S、L55R、A57V)并檢測(cè)它們對(duì)U937細(xì)胞的增殖影響,結(jié)果顯示除A57V之外,其余3個(gè)突變體均失去抑制U937細(xì)胞增殖的活性,這3個(gè)突變體除R49S與肝素結(jié)合相關(guān)外,其余2個(gè)均與肝素結(jié)合無(wú)關(guān),說(shuō)明PF4抑制U937細(xì)胞生長(zhǎng)與第37、38、39、49、55位的氨基酸密切相關(guān),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(xì)胞增殖可能與第三種機(jī)理相關(guān)。2003年,Lasagni等首次報(bào)道了PF4的唯一受體CXCR3—B,PF4與CXCR3—B結(jié)合,可抑制內(nèi)皮細(xì)胞的增殖,為研究PF4抑制腫瘤生長(zhǎng)的機(jī)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(xì)胞增殖的可能的作用機(jī)理。首先制備了兔/鼠抗人CXCR3—B分子的多/單克隆抗體,并通過(guò)三個(gè)不同區(qū)段的多肽對(duì)單克隆抗體的抗原表位進(jìn)行了初步的篩選。共獲得三種針對(duì)不同區(qū)段的多肽的多/單克隆抗體,這些抗體為進(jìn)行有關(guān)PF4抑制U937細(xì)胞增殖的作用機(jī)理研究奠定了基礎(chǔ)。第二,通過(guò)RT—PCR、免疫印跡及免疫沉淀的方法證實(shí)U937細(xì)胞表達(dá)CXCR3—B分子,Pull-down、流式細(xì)胞術(shù)的結(jié)果證明PF4可識(shí)別CXCR3—B分子并與之結(jié)合,說(shuō)明PF4抑制U937細(xì)胞的增殖可能是通過(guò)CXCR3—B介導(dǎo)的。第三,通過(guò)蛋白組學(xué)和基因芯片的實(shí)驗(yàn)技術(shù)對(duì)PF4作用于U937細(xì)胞后的蛋白及表達(dá)變化基因進(jìn)行了初步的篩選。結(jié)果顯示PF4可從U937細(xì)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(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392
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本文編號(hào)：1875542