【摘要】： 內(nèi)皮祖細(xì)胞(endothelial progenitor cells,EPCs)由骨髓釋放到外周血,參與病損傷區(qū)域的血管新生是治療血管生成相關(guān)疾病的重要途徑。EPCs從骨髓中動(dòng)員、遷移、歸巢到血管新生部位,參與血管生成過程中受到外源信號(hào)和內(nèi)在因素的級(jí)聯(lián)動(dòng)態(tài)調(diào)節(jié)�；|(zhì)細(xì)胞衍生因子-1(Stromal cell-derived factor-1,SDF-1)就是一種重要的細(xì)胞的外源信號(hào)趨化因子,與其受體結(jié)合后能調(diào)控許多重要的生物反應(yīng)過程。CXCR7(chemokine (C-X-C motif) receptor 7 )是近年來發(fā)現(xiàn)的SDF-1的新受體,但是SDF-1/ CXCR7是否參與調(diào)控血管生成過程國內(nèi)外的相關(guān)文獻(xiàn)報(bào)道幾乎沒有。本研究以EPCs為切入點(diǎn),體外考察SDF-1/ CXCR7對(duì)內(nèi)皮祖細(xì)胞增殖、遷移、黏附和凋亡過程的影響,探討CXCR7在SDF-1調(diào)控EPCs動(dòng)員、歸巢過程中扮演的角色,初步探索其作用機(jī)理,以便論證以CXCR7為靶標(biāo)進(jìn)行促進(jìn)或抑制血管生成的可能性,以期為血管新生的診斷及相關(guān)疾病的治療提供新的理論依據(jù)。本文的主要研究內(nèi)容和結(jié)果如下: ①用密度梯度離心法分離大鼠骨髓單個(gè)核細(xì)胞,經(jīng)EBM-2培養(yǎng)基培養(yǎng)呈現(xiàn)“星型集落”等典型結(jié)構(gòu),通過內(nèi)皮祖細(xì)胞特異性標(biāo)志CD31和VE-cadherin以及Ac-LDL和UEA-1鑒定,證明骨髓來源的單個(gè)核細(xì)胞可體外誘導(dǎo)成為功能性的EPCs。 ②用Reverse transcription-PCR和western-blot免疫印跡法檢測(cè)EPCs細(xì)胞中CXCR4(chemokine (C-X-C motif) receptor 4)和CXCR7的表達(dá)情況;用MTT法測(cè)定分別用抗體阻斷CXCR4、CXCR7后,對(duì)EPCs細(xì)胞增殖率的影響;通過transwel小室體外侵襲遷移實(shí)驗(yàn)檢測(cè)anti-CXCR4和anti-CXCR7對(duì)EPCs細(xì)胞趨化活性的影響。用CXCR4和CXCR7抗體分別預(yù)處理EPCs細(xì)胞后,考察其與單層HUVEC細(xì)胞黏附作用;通過流式細(xì)胞儀檢測(cè)anti-CXCR4和anti-CXCR7對(duì)經(jīng)低濃度雙氧水預(yù)處理后EPCs細(xì)胞活性的影響。結(jié)果表明:EPCs細(xì)胞中都表達(dá)CXCR4和CXCR7這兩個(gè)受體。用CXCR7抗體封閉后,EPCs細(xì)胞的增殖活力顯著下降。CXCR7抗體對(duì)SDF-1誘導(dǎo)的EPCs細(xì)胞遷移影響不顯著。CXCR4與CXCR7抗體都會(huì)抑制EPCs細(xì)胞與HUVEC細(xì)胞單層的黏附,CXCR7抗體抑制效果更明顯。SDF-1主要通過CXCR7有效保護(hù)EPCs,抑制EPCs凋亡。 結(jié)論:SDF-1對(duì)內(nèi)皮祖細(xì)胞的增殖、遷移、黏附和凋亡有直接的促進(jìn)作用,CXCR7在其中起著與CXCR4不完全相同、但相互協(xié)同的重要作用�？贵w阻斷CXCR7能夠抑制EPCs細(xì)胞的增殖、遷移、粘附和凋亡,表明CXCR7有作為心血管疾病治療靶標(biāo)的潛力。
[Abstract]:Endothelial progenitor cells (endothelial progenitor cells) are released from bone marrow to peripheral blood. Angiogenesis involved in the injured area is an important way to treat angiogenic diseases. EPCs mobilize from bone marrow, migrate and homing to angiogenesis site. Involved in angiogenesis is dynamically regulated by exogenous signals and internal factors. Stromal cell-derived factor-1 (Stromal cell-derived factor-1 SDF-1) is an important exogenous signal chemokine in cells. After binding to its receptor, it can regulate many important biological reaction processes. CXCR7 (chemokine (C-X-C motif) receptor 7 is a new receptor of SDF-1 which has been discovered in recent years. However, there are few reports on whether SDF-1/ CXCR7 is involved in the regulation of angiogenesis at home and abroad. In this study, we investigated the effects of EPCs on the proliferation, migration, adhesion and apoptosis of endothelial progenitor cells (EPCs) in vitro, explored the role of CXCR7 in the process of EPCs mobilization and homing by SDF-1, and explored its mechanism. In order to demonstrate the possibility of promoting or inhibiting angiogenesis with CXCR7 as the target, it can provide a new theoretical basis for the diagnosis of angiogenesis and the treatment of related diseases. The main contents and results are as follows: 1 the mononuclear cells of rat bone marrow were isolated by density gradient centrifugation. The mononuclear cells were cultured on EBM-2 medium with typical structure such as "star colony". The specific markers of endothelial progenitor cells (CD31, VE-cadherin, Ac-LDL and UEA-1) were identified. The results showed that mononuclear cells derived from bone marrow could be induced into functional EPCs.2 the expression of CXCR4 (chemokine (C-X-C motif) receptor 4 and CXCR7 in EPCs cells was detected by Reverse transcription-PCR and western-blot Western blotting, the expression of CXCR4 (chemokine (C-X-C motif) receptor 4 and CXCR7 in EPCs cells was detected by MTT assay after blocking CXCR4CXCR7 with antibodies. The effect of anti-CXCR4 and anti-CXCR7 on the chemotaxis of EPCs cells was detected by transwel chamber invasion and migration in vitro. The adhesion of EPCs cells to monolayer HUVEC cells was investigated after pretreatment with CXCR4 and CXCR7 antibodies, and the effects of anti-CXCR4 and anti-CXCR7 on EPCs cell activity after pretreatment with low concentration of hydrogen peroxide were detected by flow cytometry. The results showed that both CXCR4 and CXCR7 receptors were expressed in the cells. After blocking with CXCR7 antibody, the proliferation activity of EPCs cells decreased significantly. CXCR7 antibody had no significant effect on the migration of EPCs cells induced by SDF-1. Both CXCR4 and CXCR7 antibody could inhibit the adhesion of EPCs cells to HUVEC cell monolayer. The inhibitory effect of CXCR7 antibody was more obvious. It is necessary to protect EPCs effectively and inhibit EPCs apoptosis through CXCR7. Conclusion the cell proliferation, migration, adhesion and apoptosis of endothelial progenitor cells were directly promoted by WSDF-1. CXCR7 plays an important role in the proliferation, migration, adhesion and apoptosis of endothelial progenitor cells, which is not exactly the same as that of CXCR4, but plays a synergistic role. Antibody blocking of CXCR7 can inhibit the proliferation, migration, adhesion and apoptosis of EPCs cells, indicating that CXCR7 has the potential as a target for the treatment of cardiovascular diseases.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

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本文編號(hào)：2140881