【作者】 喻杨；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 1.背景与目的:血管内皮损伤是经皮冠脉介入术(percutaneous coronary intervention, PCI)血管再狭窄及支架内血栓形成的核心[1],也是动脉粥样硬化、高血压、糖尿病等多种血管性疾病共同的病理生理基础[2-4]。内皮的有效再生是抑制血管平滑肌的异常增生和防止血栓形成的关键[5]。因此,尽早促进受损血管再内皮化、恢复内皮功能,俨然是抑制血管损伤不良修复,预防或防治血管再狭窄及血栓形成的有效策略。传统观念认为内皮损伤的修复仅仅依靠邻近内皮细胞的迁移和增殖。近年的大量研究发现内皮祖细胞(endothelial progenitor cells, EPCs)是机体促进内皮有效再生以及血管良性修复的重要内在机制[6-9]。现已证明,EPCs在体内微环境的作用下能够分化成有功能的内皮细胞,参与缺血组织的血管新生并整合到损伤血管壁的新生内膜中参与损伤血管的再内皮化[10-13]。然而,目前对于调控EPCs增殖、迁移和分化的机制及某些关键因子在其中的作用尚不完全清楚。CCN1是近年发现的一种分泌型基质信号蛋白[14],广泛参与分化、发育、肿瘤生长等多种病理生理过程的调控[15-18]。已有研究表明:1)胚胎发育过程中CCN1基因缺陷将导致胎盘血管生成减少以及分支障碍,引起胚胎死亡[21]。2)成年后CCN1的表达与血管性疾病如动脉粥样硬化、原发性高血压、肿瘤血管新生等密切相关[22-25];CCN1还参与了皮肤、骨骼、肝脏等多种组织的再生修复过程[26-28]。3)在体外,CCN1能促进大鼠角膜及兔股动脉缺血后的血管新生,并对血管内皮细胞的增殖、迁移及管样形成能力均有促进作用[29-33]。4) CCN1在骨髓间充质干细胞(MSCs)的增殖、分化、迁移中发挥重要调控作用,并诱导CD34+细胞的迁移和归巢[33-35]。提示:CCN1可能在EPCs介导的血管新生及血管损伤修复中发挥重要作用。因此在本课题中,我们将从整体动物和和细胞水平探讨CCN1在EPCs增殖、迁移、分化以及血管损伤修复中的作用及其机制。期望为深入认识CCN1的生物学功能及EPCs的调控机制提供新的实验依据,为进一步促进血管损伤后内皮再生、血管良性修复提供新的思路和策略。2.方法:2.1重组腺病毒Ad-CCN1和Ad-Id1的构建从大鼠组织提取RNA,经RT-PCR扩增得到目的基因CCN1和Id1片段,通过pMD19T-Simple和AdEasy细菌内同源重组系统构建CCN1和Id1的病毒过表达载体,分别命名为Ad-CCN1和Ad-Id1,经过测序、PCR和酶切鉴定重组病毒Ad-CCN1和Ad-Id1的构建。2.2观察CCN1在血管损伤修复过程中的表达及作用用1-2月龄的雄性SD大鼠复制颈动脉球囊损伤模型,通过荧光定量RT-PCR以及Western blot分别观察CCN1在血管损伤修复过程中mRNA和蛋白表达水平的变化;然后向损伤血管组织转染Ad-CCN1,观察过表达CCN1对损伤后4、7、14、21天血管再内皮化以及新生内膜增厚的影响。2.3 CCN1对EPCs增殖、迁移和分化的作用通过密度梯度离心及选择性培养的方法在体外分离、培养大鼠骨髓源EPCs,经过细胞形态学、表面分子标志以及Dil-acLDL/FITC-UEA-I双阳性鉴定后,转染Ad-CCN1以及含CCN1 shRNA的重组质粒pGenesil-CCN1,以观察过表达或沉默CCN1基因对EPCs增殖、迁移、分化的作用。2.4探讨CCN1作用于EPCs增殖、迁移和分化的分子机制2.4.1利用GEArray公司的DNA芯片初步分析Ad-CCN1作用后48小时对EPCs基因表达的影响,筛选CCN1作用的可能下游靶点;2.4.2检测Ad-CCN1作用下负性转录调控因子Id1在转录、蛋白表达水平的变化,并通过Ad-Id1与Ad-CCN1的共转染实验观察Id1对Ad-CCN1诱导的EPCs分化的影响,以明确Id1是否参与介导Ad-CCN1对EPCs分化的调控作用。3.结果:3.1.重组腺病毒Ad-CCN1和Ad-Id1的构建从大鼠组织提取RNA进行RT-PCR获得含酶切位点的CCN1和Id1基因全CDS区,将目的基因连接到pMD19T-Simple载体中扩增,经过酶切、连接、转化等步骤后获得pAdTrack-CCN1和pAdTrack-Id1,然后与骨架质粒pAdEasy-1在BJ5183细菌内同源重组,通过筛选、293T细胞包装后获得重组病毒Ad-CCN1和Ad-Id1,经鉴定、扩增获得高滴度的Ad-CCN1和Ad-Id1病毒颗粒用于下一步实验。Ad-CCN1和Ad-Id1的病毒滴度约为1.2×1010 -2.8×1011 pfu/ml。3.2 CCN1在血管损伤修复中的表达及作用3.2.1大鼠颈动脉损伤模型的建立和转染经损伤血管组织切片H&E染色证实,我们成功复制了大鼠颈动脉球囊损伤模型,损伤后7天开始出现镜下可见的内膜增生,21天时新生内膜增生明显。荧光倒置显微镜下可见转染后血管组织切片中有绿色荧光表达,证实在体腺病毒转染是成功有效的,且未发现对实验动物有明显毒副作用。3.2.2 CCN1在血管损伤修复中的表达免疫组化实验显示CCN1在损伤血管的新生内膜、中膜以及外膜组织均有表达。CCN1mRNA在正常血管组织低表达,血管损伤后表达迅速上升,12h即达到高峰,之后逐渐下降,损伤后第21天仍较对照组高。Western blot检测到CCN1蛋白表达在血管损伤后第12h开始上调,24h时达到高峰,然后逐渐回落,在损伤后第14天再次达到一个较低的表达峰值,损伤后第21天仍有表达。3.2.3过表达CCN1对球囊损伤后血管再内皮化的影响通过伊文氏蓝染色观察发现,假手术对照组内皮无损伤,内皮覆盖率为100%,损伤组残留内皮覆盖率为0%,说明内皮剥脱完全。CCN1过表达明显促进第4天、7天和14天时损伤血管的再内皮化,与相同时间点Ad-GFP转染对照组比较均有显著差异(P<0.05),说明CCN1对损伤血管的再内皮化有促进作用。3.2.4过表达CCN1对球囊损伤后血管内膜增生的影响在血管损伤后第14天,Ad-CCN1转染组血管组织内、中膜比值为0.26±0.12,明显低于Ad-GFP对照组的内、中膜比值0.58±0.18(P<0.01),而两组中膜面积无明显差异(P>0.05),提示过表达CCN1抑制了球囊损伤后第14天大鼠颈动脉新生内膜的增厚。在损伤后第21天,CCN1过表达组与GFP对照组比较无明显差异(1.13±0.21 vs 1.20±0.19,P>0.05),说明损伤后第21天CCN1抑制血管新生内膜增厚的作用不明显。3.3 CCN1对EPCs增殖、迁移和分化的作用3.3.1 EPCs鉴定分离培养的EPCs经过诱导分化后向内皮细胞表型转变,流式细胞检测CD34、CD133及VEGFR-2在培养细胞中的阳性率分别为90.28%、93.86%和74.03%,而造血细胞表面抗原CD45的阳性表达率则十分低(<10%),Dil-acLDL/FITC- UEA-I双阳性细胞约占90%,说明所培养细胞是EPCs。3.3.2基因转染EPCs转染后细胞状态良好,贴壁生长,无变圆、缩小或脱落等病理迹象,经过荧光倒置显微镜、RT-PCR以及Western blot观察发现Ad-CCN1的转染效率在第24小时约60%,48-72小时达高峰,约80%。pGenesil-CCN1转染后4天, CCN1在EPCs的表达被显著抑制,转染率约50%。3.3.3 Ad-CCN1对EPCs增殖的影响采用MTT法分析发现,Ad-CCN1对EPCs增殖没有显著作用。虽然与Ad-GFP对照组比较,Ad-CCN1对EPCs的增殖有明显促进作用(* P<0.05),但与未转染对照组比较发现,Ad-CCN1对EPCs增殖的作用没有统计学差异(P>0.05)。3.3.4 Ad-CCN1对EPCs迁移的影响外源性CCN1的过表达显著促进了EPCs的迁移。在Ad-CCN1作用诱导下,平均每个视野中迁移EPCs的数目从7.1±1.8增加到26.1±2.8,增加了近3倍(* P<0.01)。加入CCN1封闭性抗体CCN1-Ab后,Ad-CCN1的作用明显减弱(# P<0.05),提示EPCs迁移能力的增强是过表达CCN1引起的。3.3.5 Ad-CCN1对EPCs分化的影响Ad-CCN1诱导细胞获得内皮样细胞形态,表现为贴壁伸展、体积增大、呈长梭形或多角形;FACS结果显示,CD34在Ad-CCN1和Ad-GFP两组的阳性表达率分别是13.83±5.23%、59.79±9.61% , VE-cadherin的阳性表达率分别是85.74±7.11%、14.04±6.28%;Dil-acLDL阳性细胞数在Ad-CCN1组为93.5±6.3%,明显高于Ad-GFP组的61.3±9.2%。3.3.6利用RNA干扰沉默CCN1对EPCs增殖、迁移和分化的作用结果表明pGenesil-CCN1介导的CCN1基因的沉默明显抑制了EPCs的增殖、迁移和分化功能,与未干预组及pGenesil空质粒对照组比较均有显著差异(* p<0.05)。3.4 CCN1作用于EPCs的分子机制探讨3.4.1 CCN1对EPCs基因表达的影响Ad-CCN1作用后48小时,EPCs中有8个基因明显上调2倍以上,包括4个生长因子(受体)Ereg、Vegf-c、Igf-1、和Kdr,2个基质相关分子Itgav和Timp2,1个细胞因子Tnf和1个信号蛋白Gna13;此外,12个基因的表达在CCN1作用下明显下调(2倍以上),也主要是生长因子和基质分子两大类相关基因,包括Vegf-b、Tgfa、Tgfb1、Mdk、Ptn、Thbs2、Timp3、plau(u-PA)、Ifn-a1、Cxcl5、Akt1以及Sh2d2a。3.4.2 Id1对CCN1促EPCs分化作用的影响通过荧光定量RT-PCR及Western blot检测发现Ad-CCN1显著降低了Id1在EPCs中的表达,p<0.05。单独转染Ad-Id1对EPCs分化无显著影响,与Ad-CCN1的共转染则显著抑制了Ad-CCN1对EPCs分化的促进作用,p<0.05,提示Id1抑制了CCN1对EPCs分化的促进作用。4.结论:4.1 CCN1在球囊损伤的大鼠颈动脉组织动态高表达;过表达CCN1显著促进大鼠球囊损伤早期血管再内皮化程度;过表达CCN1抑制大鼠球囊损伤第14天血管新生内膜的增厚;4.2过表达CCN1基因促进EPCs的迁移和分化;沉默CCN1基因抑制EPCs的增殖、迁移和分化;4.3过表达CCN1诱导EPCs中20个基因表达的显著变化;4.4过表达CCN1抑制Id1在EPCs的表达;Id1至少部分抑制CCN1对EPCs分化的促进作用。更多还原

【Abstract】 1. Background and Objective:Percutaneous coronary intervention (PCI) is widely used in the treatment of coronary artery disease. Despite the advent of drug-eluting stent that is promising in reducing the incidence of restenosis, it remains a considerable clinical challenge. Endothelial progenitor cells (EPCs), which can differentiate into mature endothelial cells (ECs), are increasingly recognized to play a key role in vascular regeneration. However, early reendothelialization by regenerated ECs is critical for reducing or preventing postangioplasty restenosis and thrombosis. It is crucial, therefore, to understand the molecular mechanism leading to EPCs differentiation and contribution to the repair process after vascular injury.CCN1, a secreted matricellular protein belonging to the CCN family, is expressed by all types of vascular cells. It is required for vascular development and is a potent regulator of angiogenesis. CCN1-deficient mice undergo embryonic lethality as a result of placental vascular insufficiency and compromised vessel integrity. Recombinant CCN1 induces angiogenesis in vivo, and stimulates ECs survival, migration and tube formation in culture. Moreover, it’s noteworthy that expression of CCN1 is up-regulated in vascular disorders, including atherosclerosis, mechanical injury, hypertension, and tumorigenesis. Besides, CCN1 has been associated with tissue self-renewal, such as bone fracture repair, liver regeneration, and cutaneous wound healing, suggesting a role for CCN1 in vascular regeneration, potentially involving circulating EPCs. However, the precise role of CCN1 in vascular regeneration remains largely unexplored. Evidence indicates that CCN1 is critically involved in the regulation of mesenchymal stem cells (MSCs) adhesion, migration, and differentiation. Recent investigation demonstrates that CCN1, in the plasma and endothelium surface, promotes the migration, adhesion, and recruitment of circulating human bone marrow-derived CD34+ progenitor cells to ECs and, thus play an important role in microenvironment-mediated biological properties of EPCs. All of these findings support the hypothesis that CCN1 may have a role in mediating EPCs functions, contributing to the neovascularization and vascular repair process after injury.In this study, we overexpressed CCN1 and siRNA to evaluate the possible role of CCN1 on EPCs proliferation, migration, differentiation, and participation in vascular regeneration. Our findings provide a novel insight into understanding of biological function of CCN1 and the molecular mechanisms behind EPCs-mediated vascular regeneration.2. Methods:2.1 Construction of recombinant adenoviral vectorsAdenoviral vectors repectively expressing CCN1 and Id1 were generated using the AdEasy system. Briefly, full-length rat CCN1 and Id1 cDNA were generated by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart and spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-CCN1 and pAdTrack-Id1. The shuttle vectors were used to generate recombinant adenoviruses according to the manufacturer’s protocol. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.2.2 Expression and function of CCN1 during vascular repair following rat carotid artery balloon angioplastyTo evaluate the role of CCN1 in vascular repair in vivo, we first examined the expression of CCN1 in balloon-injured rat carotid artery, using real-time quantitative PCR and Wester blot analysis. Next, adenoviral vectors expressing exogenous CCN1 or GFP as control were constructed and injected into balloon-injured artery to investigate CCN1 gene function during vascular injury. Overexpression of CCN1 was confirmed by immunohistochemistry. No observable adverse side effect (mortality or any other clinical signs of distress/morbidity) was found in experimental animals. Evans Blue dye was administered to evaluate reendothelialization at 4, 7, and 14 days after injury, and the neointimal formation was assessed at 14 and 21 days following vascular injury.2.3 Effecs of CCN1 on EPCs proliferation, migration, and differentiation EPCs were isolated by density gradient centrifugation and cultured in low glucose DMEM supplemented with 10% FCS and 10ng/mL VEGF. To confirm the EPCs phenotype, cells were incubated with DiI-acLDL for 4 hours, fixed with 4% paraformaldehyde and then incubated with FITC-labeled lectin (UEA-1) for 1 hour. Dual-stained cells positive for both DiI-acLDL and UEA-1 were identified as EPCs. Additionally, flow cytometry (FACS) analysis was performed using antibodies against rat CD45, CD133, CD34, and VEGFR-2. To investigate the effect of CCN1 on EPCs migaration, proliferation, and differentiation in vitro, we transduced Ad-CCN1 and pGenesil-CCN1 into EPCs that were cultured in serum- and VEGF- free medium.2.4 Molecular mechanisms underlying CCN1 effects on EPCsTo identify the molecular mechanism that might be involved in the effect of CCN1 on EPCs, we performed microarray analysis on EPCs stimulated with CCN1 or the GFP control, using GEArray. Genes that were either up-regulated or down-regulated 2-fold or more were determined. Moreover, we next examined the effect of CCN1 on Id1 expression, and overexpressed Id1 in EPCs to further confirm the impact of Id1 in CCN1-induced differentiation of EPCs.3. Results:3.1 Recombinant adenoviral vectors expressing CCN1 or Id1Full length cDNA encoding either CCN1 or Id1 was amplified by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart or spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into adenoviral shuttle vector pAdTrack-CMV. Recombinant adenovirus Ad-CCN1 and Ad-Id1 were generated and purified according to the manufacturer’s protocol. The adenovirus virus titer was about 1.2×1010 -2.8×1011 plaque-forming units per millilitre (pfu /ml), as determined by plaque assay.3.2 Expression and function of CCN1 during vascular repair following rat carotid artery balloon angioplasty3.2.1 Rat carotid balloon-injury modelHistological analysis and H&E staining demonstrated that neointimal formation was initiated at 7days and obviously developed at 21day after balloon-mediated vascular injury in control rats. In addition, after transfection with Ad-CCN1 or Ad-GFP, green fluorescence was detected with vascular tissues under a fluorescence microscope at 48 h post-vascular injury, indicating the efficiency of adenovirus transfection in rat carotid arteries.3.2.2 Expression of CCN1 during vascular repair processCCN1 mRNA expression was detected at low levels in normal uninjured control arteries, whereas following vascular injury CCN1 mRNA level was rapidly enhanced with a peak at 12 h which gradually declined thereafter in 4 days and remained elevated for up to at least 21days. CCN1 protein expression was assessed by Western blotting and was consistently found to be up-regulated. Further, immunohistochemistry showed that CCN1 was detected in the intima, media, and adventitia of local vessels.3.2.3 Effect of Ad-CCN1 on vascular reendothelializationEvans Blue dye was administered to evaluate reendothelialization at 4, 7 and 14 days after injury. Nonendothelialized lesions were marked blue about 100% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. At all time points, the reendothelialized area in the Ad-CCN1-infected arteries was significantly larger than that in Ad-GFP infected arteries (p<0.05).3.2.4 Effect of Ad-CCN1 on neointimal formationA marked decrease in the neointimal area and I/M ratio (0.26±0.12 vs 0.58±0.18, p<0.01) was shown in Ad-CCN1-treated rats compared with that of control group at day 14. However, no difference was found at 21 days (1.13±0.21 vs 1.20±0.19, p>0.05), suggesting that CCN1 effectively prevented neointimal hyperplasia at early stage.3.3 Effecs of CCN1 on EPCs proliferation, migration, and differentiation3.3.1 EPCs isolation and characterizationAfter 4-7 days of culture, adherent EPCs were characterized by immunofluorescence and flow cytometry analysis (FACS). The majority of cells (>90%) stained positive for DiI-AcLDL and lectin, and expressed endothelial/stem cell markers, including CD34 (90.28%), VEGFR-2 (74.03 %), and CD133 (93.86 %), but not hematopoietic cell marker CD45 (<10 %.), confirming the cell type of EPCs.3.3.2 EPCs transfectionAdenovirus-mediated CCN1 expression was confirmed by fluorescence, RT-PCR, and Western blot analysis. The transfection efficiency was 60% at 24h post-transfection and 80% at 48-72h post-transfection. Four days after introduction of pGenesil1-CCN1, an approximate 50% of CCN1 expression loss was shown, as measured by Western blot and RT-PCR.3.3.3 Effect of Ad-CCN1 on EPCs proliferationThe proliferation of EPCs was not ehanced significantly by Ad-CCN1. Despite a decrease observed in cells transfected with Ad-CCN1 as compared with Ad-GFP (p<0.05), it has no significant meaning in compare with untransfected control (p>0.05). Our findings suggested that adenovirus maight inibit the proliferation of EPCs.3.3.4 Effect of Ad-CCN1 on EPCs migrationOver expression of exogenous CCN1 extensively improved the migration of EPCs, and in fact, the average migrated cell number of the EPCs increased by approximately 3-fold, from 7.1±1.8 to 6.1±2.8 (* P<0.01) compared to that of the control cells.3.3.5 Effect of Ad-CCN1 on EPCs differentiationAfter Ad-CCN1 infection, a noticeable amount of EPCs transformed into a spindle-or polygonal-shaped appearance, which was less observed in control Ad-GFP transduced EPCs. Additionally, the expression of the progenitor marker CD34 was drastically decreased (13.83±5.23% in Ad-CCN1 group vs 59.79±9.61% in Ad-GFP group), whereas the expression of ECs marker VE-cadherin was increased conversely (85.74±7.11% in Ad-CCN1 group vs 14.04±6.28% in Ad-GFP group). Immunofluorescence study showed that the percentage of DiI-acLDL-positive cells was significantly increased in Ad-CCN1 group (93.5±6.3% vs 61.3±9.2%, p<0.05).3.3.6 Role of siRNA-CCN1After introduction of pGenesil1-CCN1, EPCs exhibited a decrease in cell proliferation, migration and differentiation when compared with negative control siRNA transfected cells or untransfected cells (* p< 0.05).3.4 Molecular mechanisms underlying CCN1 effects on EPCs3.4.1 Microarray analysisWe found that at 48 hours upon Ad-CCN1 stimulation, 8 genes were up-regulated in EPCs compared with Ad-GFP controls. Among these were prominent growth factor and receptor genes such as Vegf-c, Kdr, Igf-1, and Ereg, as well as Tnf, Timp2, Itgav, and Gna13. In contrast, 12 genes were significantly down-regulated in EPCs transfected with Ad-CCN1. These genes include Vegf-b, Tgfα, Tgfβ, Mdk, Ptn, Ifn-α1, Cxcl5, Timp3, Thbs, Plau, Akt1, and Sh2d2a, most of which were corresponding to growth factors, receptors, chemokines, and adhesion molecules, pointing to their potential involvement in the effects of CCN1 on EPCs.3.4.2 Role of transcription factor Id1In response to Ad-CCN1, a decrease of Id1 mRNA expression was detected in EPCs at 48 hours (p<0.05). Transfection with Ad-Id1 alone induced no apparent changes in Dil-acLDL positive cells and expression of cell markers compared with that of Ad-GFP. However, cotransfection with Ad-CCN1 and Ad-Id1 significantly inhibited the Ad-CCN1 induced differentiation of EPCs, p<0.05.4. Conclusions:4.1 CCN1 was dynamically expressed in vascular lesions and found to promote reendothelialization and inhibit neointimal formation at the early stage after vascular injury;4.2 Overexpression of CCN1 stimulated EPCs migration and differentiation and that was reversed by siRNA-mediated silencing of CCN1 expression;4.3 There were 8 genes up-regulated and 12 genes down-regulated in EPCs upon Ad-CCN1 stimulation, suggesting their potential involvement in the mechanism of CCN1 effect on EPCs;4.4 Ad-CCN1 inhibited the expression of Id1 in EPCs, and Id1, at least partly, inhibited CCN1-induced differentiation of EPCs.更多还原