基质细胞衍生因子（SDF-1）的新受体CXCR7对内皮祖细胞调节作用的初探
发布时间：2018-07-24 09:01
【摘要】： 内皮祖细胞(endothelial progenitor cells,EPCs)由骨髓释放到外周血,参与病损伤区域的血管新生是治疗血管生成相关疾病的重要途径。EPCs从骨髓中动员、迁移、归巢到血管新生部位,参与血管生成过程中受到外源信号和内在因素的级联动态调节。基质细胞衍生因子-1(Stromal cell-derived factor-1,SDF-1)就是一种重要的细胞的外源信号趋化因子,与其受体结合后能调控许多重要的生物反应过程。CXCR7(chemokine (C-X-C motif) receptor 7 )是近年来发现的SDF-1的新受体,但是SDF-1/ CXCR7是否参与调控血管生成过程国内外的相关文献报道几乎没有。本研究以EPCs为切入点,体外考察SDF-1/ CXCR7对内皮祖细胞增殖、迁移、黏附和凋亡过程的影响,探讨CXCR7在SDF-1调控EPCs动员、归巢过程中扮演的角色,初步探索其作用机理,以便论证以CXCR7为靶标进行促进或抑制血管生成的可能性,以期为血管新生的诊断及相关疾病的治疗提供新的理论依据。本文的主要研究内容和结果如下: ①用密度梯度离心法分离大鼠骨髓单个核细胞,经EBM-2培养基培养呈现“星型集落”等典型结构,通过内皮祖细胞特异性标志CD31和VE-cadherin以及Ac-LDL和UEA-1鉴定,证明骨髓来源的单个核细胞可体外诱导成为功能性的EPCs。 ②用Reverse transcription-PCR和western-blot免疫印迹法检测EPCs细胞中CXCR4(chemokine (C-X-C motif) receptor 4)和CXCR7的表达情况;用MTT法测定分别用抗体阻断CXCR4、CXCR7后,对EPCs细胞增殖率的影响;通过transwel小室体外侵袭迁移实验检测anti-CXCR4和anti-CXCR7对EPCs细胞趋化活性的影响。用CXCR4和CXCR7抗体分别预处理EPCs细胞后,考察其与单层HUVEC细胞黏附作用;通过流式细胞仪检测anti-CXCR4和anti-CXCR7对经低浓度双氧水预处理后EPCs细胞活性的影响。结果表明:EPCs细胞中都表达CXCR4和CXCR7这两个受体。用CXCR7抗体封闭后,EPCs细胞的增殖活力显著下降。CXCR7抗体对SDF-1诱导的EPCs细胞迁移影响不显著。CXCR4与CXCR7抗体都会抑制EPCs细胞与HUVEC细胞单层的黏附,CXCR7抗体抑制效果更明显。SDF-1主要通过CXCR7有效保护EPCs,抑制EPCs凋亡。 结论:SDF-1对内皮祖细胞的增殖、迁移、黏附和凋亡有直接的促进作用,CXCR7在其中起着与CXCR4不完全相同、但相互协同的重要作用。抗体阻断CXCR7能够抑制EPCs细胞的增殖、迁移、粘附和凋亡,表明CXCR7有作为心血管疾病治疗靶标的潜力。
[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.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
本文编号：2140881
[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.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
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