【作者】 尹扬光；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 1.背景与目的:冠心病是目前导致死亡的重要原因。经皮冠脉介入(Percutaneous coronary intervention, PCI)血运重建有效改善冠心病患者预后,成为本病的主要治疗手段之一。PCI术后血管再狭窄、血栓形成及血管痉挛是导致心脏事件的主要原因。药物洗脱支架的应用降低了PCI术后再狭窄率,却未降低血栓形成及血管痉挛的发生率。目前,PCI术后并发症的防治仍是心血管领域研究的热点之一。PCI造成靶血管段机械损伤,内皮细胞剥脱,成为局部平滑肌细胞(Smooth muscle cells,SMC)迁移和增生的基础;药物洗脱支架释放的细胞毒药物雷帕霉素或紫杉醇,有效抑制平滑肌细胞SMC迁移和增生,诱导其凋亡。但细胞毒药物同时也抑制内皮细胞的迁移和增生,也诱导内皮细胞凋亡,对损伤血管的再内皮化(Reendothelialization)过程具有明显的抑制作用。损伤血管无内皮覆盖促进SMC迁移和增生,内皮下胶原暴露激活血液中凝血系统促进损伤血管局部形成血栓。此外,无内皮覆盖的血管生成一氧化氮(NO)和前列腺环素等血管保护性细胞因子的能力明显降低,是导致PCI术后血管再狭窄、血栓形成及血管痉挛的另一个重要原因。因此,及时再内皮化损伤血管可抑制SMC迁移和增生,阻断凝血系统的激活,恢复血管保护性细胞因子的生成,在PCI术后并发症的预防中起关键作用。以往认为,血管损伤后内皮细胞剥脱,损伤局部边缘的内皮细胞增殖、迁移是再内皮化损伤血管的唯一内皮细胞来源。系列的研究表明,骨髓或外周血源性的内皮祖细胞(Endothelial progenitor cells,EPCs)在体内局部微环境的作用下能够分化成有功能的内皮细胞,整合到损伤血管壁的新生内皮中参与损伤血管的再内皮化。采用药物、细胞因子和激素等可显著动员骨髓源性EPCs进入外周血,起到加速损伤血管再内皮化,抑制新生内膜增生的作用。移植的EPCs可成功募集到损伤血管局部分化成内皮细胞参与并加速损伤血管的再内皮化,抑制新生内膜增生。在EPCs介导的缺血组织血管新生的研究中发现,趋化因子基质细胞衍生因子-1(Stromal cell derived factor-1,SDF-1,又称CXCL12)参与缺血诱导的EPCs动员及参与介导EPCs在缺血组织中归巢。在Schober和Zernecke等的研究中发现,小鼠颈动脉损伤后损伤血管局部SDF-1表达明显上调,这种血管损伤后表达上调的SDF-1能够诱导骨髓Sca-1+lineage-祖细胞动员到外周血,并能够介导自体动员的或移植的Sca-1+lineage-祖细胞募集到血管损伤处分化成SMC参与新生内膜形成。然而,这种血管损伤后表达上调的SDF-1是否也能够诱导EPCs动员到外周血,是否也能够介导EPCs募集到血管损伤处分化成内皮细胞参与损伤血管的再内皮化,目前还未见相关报道。为此,本研究在体外实验部分观察SDF-1对体外扩增的EPCs增殖、迁移及凋亡的影响;在动物实验部分观察小鼠颈动脉损伤后损伤血管局部SDF-1的表达变化,外周血及骨髓中SDF-1的浓度变化,同时观察血管损伤后外周血EPCs数量的改变;在动物实验部分还观察小鼠颈动脉损伤后损伤血管局部表达上调的SDF-1是否参与介导荧光标记的EPCs募集到血管损伤处,分化成内皮细胞参与损伤血管的再内皮化,抑制新生内膜的增生。2.方法本课题第一部分采用内皮细胞选择性培养基培养和诱导分化密度梯度离心法获得的小鼠骨髓源性单个核细胞,以获得小鼠骨髓源性EPCs,并通过acLDL-DiI和FITC-lectin荧光双染鉴定、流式细胞仪检测细胞表面干细胞抗原及内皮细胞特异性抗原鉴定和免疫组化法鉴定细胞表面内皮细胞特异性抗原的表达。小鼠骨髓源性EPCs经各种浓度的SDF-1α处理,采用acLDL-DiI和FITC-lectin荧光双阳性细胞计数、MTT分析、黏附细胞计数、TUNEL法染色及改良的Boyden小室分析来分别检测SDF-1α对EPCs数量、增殖及黏附能力和抗凋亡能力的影响,及检测SDF-1α对EPCs的诱导迁移能力。本课题第二部分采用非显微外科手术方法建立小鼠颈动脉损伤模型,并应用扫描电镜及病理学方法检测小鼠颈动脉损伤模型是否建立成功。在小鼠颈动脉损伤后各时间点(0天、1天、3天、7天和14天)获取损伤侧颈总动脉,采用RT-PCR和Western blot检测损伤动脉SDF-1αmRNA表达及SDF-1α蛋白表达;在小鼠颈动脉损伤后各时间点(0天、1天、3天、7天和14天)获取小鼠外周血和骨髓,采用ELISA测定外周血和骨髓SDF-1α浓度,采用流式细胞仪检测外周血EPCs数量。采用抗SDF-1α单克隆抗体或IgG1同型对照注射颈动脉损伤小鼠,流式细胞仪检测动脉损伤后各时间点(0天、1天、3天、7天和14天)外周血EPCs数量,14天后通过Evans blue染色检测损伤动脉再内皮化情况及通过病理学方法检测损伤动脉内膜增生情况。本课题第三部分采用改良的Boyden小室分析检测AMD3100(CXCR4的特异性阻断剂)对SDF-1α诱导EPCs迁移的阻断作用。采用acLDL-DiI标记的EPCs移植、CXCR4阻断的EPCs移植及无菌生理盐水注射颈动脉损伤小鼠,14天后取小鼠损伤颈动脉,纵形剖开后荧光显微镜下观察并计数acLDL-DiI阳性的EPCs在损伤动脉壁的归巢情况;采用活体注射FITC-lectin后获取小鼠损伤颈动脉,冰冻切片荧光显微镜下观察移植的细胞参与损伤动脉再内皮化情况;采用活体注射Evans blue后获取小鼠损伤颈动脉,显微镜下观察细胞移植对损伤动脉再内皮化面积的影响;通过病理学方法检测细胞移植对损伤动脉新生内膜增生的影响。3.结果第一部分:培养7天的小鼠骨髓单个核细胞呈梭形或纺锤状,与内皮细胞形态相似,绝大多数细胞可摄取acLDL-DiI和与lectin结合,流式细胞仪检测显示这些细胞既有干细胞抗原Sca-1的高表达,又有内皮特异性标记物VEGFR-2的高表达,免疫组化检测显示这些细胞还高表达内皮特异性标记物CD31、eNOS和vWF。荧光双染计数显示,SDF-1α刺激浓度依赖性增加小鼠骨髓单个核细胞分化成EPCs的数量;MTT分析显示,SDF-1α刺激浓度依赖性增强EPCs的增殖能力;黏附细胞计数显示,SDF-1α刺激浓度依赖性增加EPCs黏附到包被有人纤维连接蛋白的细胞培养板上的数量;TUNEL法染色显示,SDF-1α刺激浓度依赖性降低紫杉醇等诱导EPCs凋亡的数量;改良的Boyden小室分析显示,SDF-1α浓度依赖性增加迁移的EPCs数量。第二部分:非显微外科手术方法损伤小鼠颈动脉后取损伤动脉扫描电镜下观察,损伤动脉内皮完全剥脱。RT-PCR分析显示,小鼠颈动脉损伤后1天、3天和7天,损伤血管局部SDF-1αmRNA表达明显增强,SDF-1αmRNA表达在动脉损伤后3天达到高峰;Western blot分析显示,小鼠颈动脉损伤后损伤血管局部SDF-1α蛋白合成也明显增加,与SDF-1αmRNA的表达增强一致。ELISA测定显示,小鼠颈动脉损伤后1天、3天和7天,外周血SDF-1α浓度明显高于假手术组(P<0.01),外周血SDF-1α浓度在动脉损伤后3天达到高峰;然而,骨髓SDF-1α浓度变化与外周血SDF-1α浓度变化恰好相反,小鼠颈动脉损伤后1天、3天和7天,骨髓SDF-1α浓度明显低于假手术组(P<0.01),骨髓SDF-1α浓度在动脉损伤后3天降至最低。流式细胞仪检测外周血EPCs数量显示,小鼠颈动脉损伤后1天、3天和7天,外周血EPCs数量明显高于假手术组(P<0.01),外周血EPCs数量在动脉损伤后3天达到高峰。SDF-1α单克隆抗体注射组小鼠仅动脉损伤后1天外周血EPCs数量明显高于假手术组(P<0.05),却明显低于IgG1同型对照组(P<0.05),动脉损伤后3天和7天外周血EPCs数量与假手术组无明显差异(P>0.05);Evans blue染色显示,SDF-1α单克隆抗体注射组小鼠损伤颈动脉再内皮化面积明显低于IgG1同型对照组(P<0.01),两组新生内膜面积及内膜与中膜的比值无明显差异(P>0.05)。第三部分:改良的Boyden小室分析显示,AMD3100孵育阻断小鼠骨髓源性EPCs表面CXCR4,可有效阻断SDF-1α诱导小鼠骨髓源性EPCs迁移。细胞移植后14天,小鼠损伤颈动脉纵形剖开荧光显微镜下观察发现,EPCs移植组小鼠损伤动脉表面黏附的acLDL-DiI阳性细胞数明显多于CXCR4阻断的EPCs移植组(P<0.01);损伤颈动脉冰冻切片置荧光显微镜下观察显示,EPCs移植组许多acLDL-DiI阳性细胞覆盖在小鼠损伤颈动脉的新生内膜表面,排列成线状,这些细胞也显示具有结合lectin的能力;CXCR4阻断的EPCs移植组损伤颈动脉新生内膜表面仅覆盖少许acLDL-DiI和lectin双阳性细胞和少许lectin单阳性细胞,未形成连续的内皮细胞线;对照组损伤颈动脉新生内膜表面仅少许lectin单阳性细胞覆盖,无连续的内皮细胞线。活体Evans blue染色后取小鼠损伤颈动脉纵形剖开显微镜下观察显示,EPCs移植组小鼠损伤颈动脉再内皮化面积明显高于对照组(P<0.01)和CXCR4阻断的EPCs移植组(P<0.01);而CXCR4阻断的EPCs移植组小鼠损伤颈动脉再内皮化面积与对照组比较无明显差异(P>0.05)。小鼠损伤颈动脉病理形态学分析显示,EPCs移植组小鼠损伤颈动脉新生内膜面积明显低于对照组(P<0.01)和CXCR4阻断的EPCs移植组(P<0.01);而CXCR4阻断的EPCs移植组小鼠损伤颈动脉新生内膜面积与对照组比较无明显差异(P>0.05)。4.结论1. SDF-1促进骨髓干细胞分化成为EPCs,且浓度依赖性增强EPCs黏附增殖能力,有效抑制紫杉醇等诱导的EPCs凋亡,SDF-1还浓度依赖性诱导EPCs趋化迁移。2.小鼠颈动脉损伤后损伤血管局部SDF-1表达上调,进而导致外周血SDF-1浓度升高,同时骨髓SDF-1表达下调,改变骨髓与外周血之间的SDF-1浓度剃度,从而诱导骨髓EPCs动员到外周血。阻断小鼠颈动脉损伤后SDF-1介导的EPCs动员,抑制损伤动脉的再内皮化过程。3.动脉损伤后损伤血管局部表达上调的SDF-1与EPCs表面CXCR4的相互作用参与介导外周血EPCs归巢到损伤动脉局部分化成内皮细胞表型,促进损伤动脉再内皮化,抑制损伤动脉新生内膜增生。4.采用内皮细胞选择性培养基培养和诱导分化密度梯度离心法获得的小鼠骨髓源性单个核细胞,可获得足量的小鼠骨髓源性EPCs供研究之用。5.采用非显微外科手术方法可成功建立小鼠颈动脉损伤模型,其优点是无需显微外科设备、器械和专业的显微外科手术人员。更多还原

【Abstract】 1. Background and Objective:Revascularization procedures such as percutaneous balloon angioplasty, stent implantation, or atherectomy are widely used in the treatment of coronary artery disease but are often prone to failure because of restenosis, thrombosis, and vasospasm. Initial endothelial denudation is a major contributing factor to these consequences, in which the availability of vascular protective molecules such as nitric oxide (NO) and prostacyclin as well as antioxidant systems such as heme oxygenase-1(HO-1) are decreased, and the production of growth-promoting substances are increased, which ultimately lead to the formation of neointima. Reendothelialization at sites of spontaneous or iatrogenic endothelial denudation has classically been thought to be the result of the migration and proliferation of endothelial cells from the viable endothelium adjacent to the injury site. Neighboring endothelial cells, however, it may not constitute the exclusive source of endothelial cells for vascular repair. Recently, a series of investigations has suggested that endothelial progenitor cells (EPCs) derived from the bone marrow are present in peripheral blood and that these cells can be recruited to denuded areas and incorporated into nascent endothelium.EPCs may be defined as adherent cells derived from peripheral blood- or bone marrow-derived mononuclear cells demonstrating ac-LDL uptake and isolectin-binding capacity. The number of circulating EPCs inversely correlates with the number of cardiovascular risk factors and is reduced in cardiovascular disease. Moreover, several studies have demonstrated that patients with coronary heart disease or severe heart failure may suffer from impaired function of peripheral circulating EPCs. EPCs mobilized or transfused systematically can home to the sites of endothelial denudation, and accelerates reendothelialization of injured arteries and effectively impairs smooth muscle cell proliferation and neointima formation. However, the means of EPCs homing to the sites of endothelial denudation is not clear yet.First clinical trials have been performed investigating the effects of EPCs transplantation into cardiac ischemic areas after myocardial infarction, in patients with peripheral atherovascular disease, and on endothelialization of artificial heart valves. Next to EPCs transplantation, the pharmacological mobilization and functional modification of EPCs may also play a major role in future therapies.Chemokine stromal cell derived factor-1 (SDF-1, also known as CXCL12) is constitutively produced by bone marrow stromal cells and by other cells including CD34+ cells. It was initially characterized as a pre-B cell-stimulating factor and believed to be involved in retention of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in bone marrow. SDF-1 has been demonstrated to increase EPCs number through enhancement of (BM) c-kit+ stem cell adhesion onto extracellular matrix components by integrin receptors and protect EPCs from serum starvation-induced apoptosis. Increasing the level of SDF-1 in perepheral blood has been shown to mobilize bone marrow-derived EPCs into peripheral blood. Up-regulated SDF-1 expression in ischemic tissues or increasing SDF-1 expression in ischemic tissues through several established methods could also mobilize bone marrow-derived EPCs into peripheral blood and mediate them home to the site of neovascularization in ischemic tissues. Recently, studies had shown that the expression of SDF-1αwas up-regulated in injured carotid arteries of apoE-/- mice, which resulted in a marked mobilization of circulating Sca-1+lineage- progenitor cells (PBPCs) in the peripheral blood and mediated these cells home to the site of neointimal lesions, where they can adopt an SMC-like phenotype. It is not clear whether the up-regulation of SDF-1αcould also induce a mobilization of EPCs and mediate them home to site of injured arteries.In the present study, we attempted to demonstrate that SDF-1 is a biological function modificator of EPCs. For this purpose, we evaluated the effects of SDF-1 on the number, proliferation, migration, adhesiveness and apoptosis of murine bone marrow-derived EPCs. To clarify the role of local SDF-1αin repair of injured artery, we investigated the effect of up-regulated SDF-1αexpression on mobilization of EPCs in peripheral blood with fluorescence-activated cell sorter analysis after wire-induced arterial injury in mice. Furthermore, spleen-derived EPCs co-cultured with AMD3100 (a highly selective antagonist of SDF-1 that binds to its receptor, CXCR4) were injected into mice after carotid injury to evaluate the effect of local SDF-1αexpression on homing of EPCs to the site of endothelial denudation.2. Methods:In part one, mononuclear cells (MNCs) in murine bone marrow were isolated by density gradient centrifugation and bone marrow-derived EPCs were cultured according to previously described techniques. Murine bone marrow-derived EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under an inverted fluorescent microscope and further documented by demonstrating the expression of Sca-1 and VEGFR-2 by flow cytometer. To investigate the potential effects of SDF-1 on EPCs biological functions, bone marrow mononuclear cells or bone marrow-derived EPCs were stimulated with different concentrations of SDF-1 (1 ng/ml,10 ng/ml or 100 ng/ml) for different times. EPCs number, proliferation, migration, adhesion and apoptosis were evaluated by counting the double positive cells, MTT assay, modified Boyden chamber assay, counting the adherent cells and TUNEL staining, respectively.In part two, we established an injury model of the mouse carotid artery with a non- microscopical surgery method. The specimens of carotid arteries 30 minutes after the denuding procedure were examined using scanning electron microscopy, and the intimal lesion 2 weeks after injury was measured with pathological method. SDF-1αwas detected by RT-PCR and Western blot in carotid arteries of mice at different time points (day 0, day 1, day 3, day 7,day14) after wire-induced injury. Peripheral blood murine samples and bone marrow were obtained from mice at different time points after induction of vascular injury or sham operation (day 0, day 1, day 3, day 7, day 14). SDF-1 determination in peripheral blood and bone marrow samples was performed by SDF-1 enzyme-linked immunosorbent assay (ELISA) kit. EPCs were quantified in peripheral blood samples by flow cytometry. EPCs were also examined in peripheral blood samples of mice in subgroups, in which blocking SDF-1 monoclonal antibody or IgG1 isotype control was injected after vascular injury. Moreover, reendothelialization was determined by Evans blue staining in a whole vessel preparation 14 days after induction of endothelial cell damage in subgroups.In part three, mice received either 1x106 Dil-Ac-LDL–labeled spleen-derived EPCs or 1x106 CXCR4-blocked EPCs by direct percutaneous intracardial injection after induction of arterial injury. Control animals received a corresponding amount of normal saline. Thirty minutes before euthanasia, mice received FITC-labeled lectin intravenously. A subset of these animals received an injection of Evans blue dye 10 minutes before tissue harvesting. Carotid arteries were harvested 14 days after wire injury. Some of them were sectioned for fluorescent microscopic analysis to investigate Dil-Ac-LDL positive cells and lectin expression. A subset of carotid arteries was opened longitudinally for en face microscopy to examine Dil-Ac-LDL positive cells or to measure the area of reendothelialization. H&E staining was performed for morphometric analyses, and Lucia Measurement software was used to measure external elastic lamina, internal elastic lamina, and lumen circumference as well as medial and neointimal area of carotid arteries in all sections.3. Results:Part one:After 7 days in endothelial cell selection medium, bone marrow mononuclear cells turned into spindle-shaped, endothelial cell-like cells. Most of them showed uptake of ac-LDL and lectin binding, demonstrating endothelial cell characteristics. These cells were characterized further by demonstrating the expression of the mouse stem-cell marker Sca-1 as well as the endothelial cell lineage antigen VEGFR-2 by flow cytometry. Endothelial cell lineage antigen CD31, eNOS and vWF were also expressed by most of these cells in immunohistochemical analysis. Counting double fluorescent staining positive cells revealed that incubation of bone marrow mononuclear cells with SDF-1αincreased the number of differentiated, adherent EPCs in a concentration-dependent manner. Data in MTT assay showed that the proliferative activity of bone marrow-derived EPCs was improved concentration-dependently by SDF-1α. Pre-exposed to SDF-1α, the adhesive activity of EPCs was increased in a concentration-dependent manner. A dose-dependent decrease of apoptosis, which was induced by paclitaxel or serum absent media, was noticed as EPCs was incubated with SDF-1α. Data revealed that SDF-1αcould mediate obvious EPCs migration in a dose-dependent manner.Part two:Complete removal of the endothelium was achieved with a non-microscopical surgery method. Detected with Scanning electron microscopy, a platelet monolayer covered the denuded surface of injured carotid arteries. 2 weeks later, obvious neointimal hyperplasia was noticed in the injured carotid arteries. The results in RT-PCR and western blotting showed that up-regulation of SDF-1αmRNA and protein were already evident at 1day, and peak expression was achieved at 3 days after arterial injury. In enzyme-linked immunosorbent assay, an obvious rise in plasmatic concentration of SDF-1αwas also noticed 1 day, 3 days and 7 days after carotid injury, compared with the sham operation group (P<0.01). In contrast, a significant reduction of SDF-1αbone marrow concentration was examined 1 day, 3 days and 7 days after carotid injury, compared with the sham operation group (P<0.01). The results in fluorescence-activated cell sorting analysis showed that the amount of circulating EPCs was increased markedly 1 day, 3 days and 7 days after induction of vascular injury, compared with the sham operation group (P<0.01). In a subgroup in which blocking SDF-1αmonoclonal antibody was injected, the amount of circulating EPCs was increased lightly only at 1 day after induction of vascular injury, compared with the sham operation group (P<0.05). Interestingly, Evans blue staining showed that the reendothelialization area in the SDF-1 mAb-treated group was reduced significantly 14 days after induction of endothelial cell damage, compared with the IgG1 isotype control group (P<0.01). Surprisingly, a reduced reendothelialization in the SDF-1 mAb-treated group was not associated with an increased neointima formation, compared with the IgG1 isotype control group (P>0.05).Part three:In modified Boyden chamber assay, pretreatment of bone marrow-derived EPCs with 10 ng/ml AMD3100 almost completely blocked cell migration in response to 100 ng/mL SDF-1α, compared with the control (P>0.05). En face microscopy 14 days after induction of injury revealed that infusion of CXCR4-free EPCs resulted a more number of cells attached at the injury site, forming islets of transplanted cells within the deendothelialized area, compared with the group treated with CXCR4-blocked EPCs (P<0.01). Immunohistochemical analysis revealed that transfused CXCR4-free EPCs were predominantly found at the injury site, lining the intraluminal margin of the neointima. Lectin staining revealed that the attaching cells at the site of endothelial injury were lectin-positive. Overlay experiments clearly demonstrated the endothelial phenotype of transfused cells. In the CXCR4-blocked EPCs transfusion group, only few Dil-Ac-LDL- and lectin-positive cells were present, and the endothelial cell line of the neointima appeared disrupted at day 14 after induction of injury compared with the CXCR4-free EPCs treatment group. In the placebo group, a few endogenous (lectin-positive, Dil-Ac-LDL–negative) endothelial cells were present at the site of endothelial denudation, and no endothelial cell line was formed. Data in Evans blue staining revealed that transfusion of CXCR4-free EPCs was associated with a significant increase in reendothelialization area compared with the placebo group (P<0.01). But no significant increase in reendothelialization area was noted in the group of CXCR4-blocked EPCs transfusion, compared with the placebo group (P>0.05). Computer-based morphometric analysis showed that the accelerated reendothelialization after transfusion CXCR4-free EPCs was associated with a significant reduction of neointima formation compared with the placebo group (P<0.01); no change in neointima formation was detected in CXCR4-blocked EPCs treated group compared with the placebo group (P>0.05).4. Conclusions:1. SDF-1αcould induce EPCs migration in a dose-dependent manner, and improved the proliferative and adhesive activity of EPCs, and inhibited EPCs apoptosis in the same manner. Moreover, SDF-1αcould induce differentiation of hematopoietic stem cells toward EPCs.2. Up-regulated SDF-1 expression after vascular injury participateded in the mobilization of EPCs through changing the gradient of SDF-1 concentration between the peripheral blood and the bone marrow. Blocking EPCs mobilization mediated by SDF-1 after vascular injury could inhibit reendothelialization of injured arteries.3. Local SDF-1 expression in injured arteries could mediate EPCs home to the site of endothelial denudation and participate in reendothelialization of neointimal lesions.4. Bone marrow-derived EPCs of mice can be obtained through culturing bone marrow mononuclear cells in endothelial cell selection medium.5. Carotid artery injury model of mouse can be established by a non-microscopical surgery method.更多还原