【作者】 李杜娟；

【导师】 阮秋蓉；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 内皮损伤或功能障碍是动脉粥样硬化和血管成形术后再狭窄等心血管疾病发生进展的始动环节。加快损伤内皮的修复可有效抑制新生内膜的增生,对动脉粥样硬化的早期预防和血管成形术后再狭窄的防治具有重要意义。近年的研究表明动员或移植的内皮祖细胞(endothelial progenitor cells, EPCs)可直接分化为内皮细胞,和或以旁分泌机制刺激成熟内皮增殖迁移,促进损伤血管的再内皮化,抑制新生内膜的增生。充足的归巢是移植EPCs功能发挥的前提,但大多数动物及临床实验显示移植EPCs的归巢和长期植入率都非常低,这也是导致治疗效果不理想的重要原因之一。事实上,EPCs在损伤区归巢聚集的数量不仅取决于循环中细胞数目也取决于移植细胞迁移粘附等生物学功能。有研究表明CXCR4的表达水平对EPCs的归巢及促内皮修复功能起重要的调控作用。然而,冠心病的危险因素可导致EPCs CXCR4表达下调或CXCR4的信号通路受损,导致EPCs的迁移归巢能力下降,严重影响了EPCs移植的疗效。因此,CXCR4表达及功能调控已成为EPCs治疗领域重要的研究课题。Foxc2蛋白是叉头框转录因子家族成员之一,对于心血管系统起重要的调控作用。在胚胎发育期,Foxc2可以调控内皮基因表达及血管的发生。近来研究表明Foxc2可通过多个环节调控血管生成。例如,Foxc2可诱导多种粘附分子和促血管生成因子的表达。此外,Foxc2可直接调控内皮细胞CXCR4的表达。然而Foxc2对EPCs生物学特性的影响,迄今尚未见报道。基于此,我们欲探讨Foxc2对EPCs CXCR4表达、归巢功能及促内皮修复效应的影响。本研究分为三个部分。第一部分小鼠骨髓内皮祖细胞的培养及鉴定目的：分离培养小鼠骨髓源内皮祖细胞并鉴定。方法：分离小鼠骨髓单个核细胞,在内皮培养系定向诱导培养15-21天,通过形态学观察细胞的生长形态,用免疫荧光、Western blot及流式细胞仪技术检测内皮标记及造血系标记,Dil-acLDL摄取及Matrigel管样结构形成实验检测内皮分化的功能学特性。结果：分离骨髓单个核细胞诱导培养14天后,培养细胞逐渐呈现单层的“铺路石样”外观。免疫荧光检测结果显示,绝大多数细胞表达内皮标记物CD31、VE-cadherin及vWF; Western blot分析进一步证实培养细胞表达内皮标记FLK-1、CD31和VE-cadherin。而流式细胞仪分析显示培养细胞几乎不表达造血系标记CD34和CD45. Dil-acLDL摄取结果显示,约95%的细胞吸收Dil-acLDL。Matrigel管状结构形成实验显示培养细胞能围成管状、网络状结构。因此,从形态学、细胞表型及功能学鉴定来看,培养细胞具有内皮集落形成细胞(endothelial colony forming cells, ECFCs)或晚期EPCs特性。结论：小鼠骨髓单个核细胞在特定诱导扩增的培养条件下可获得EPCs。第二部分Foxc2过表达对内皮祖细胞迁移及粘附能力的影响目的：研究Foxc2过表达对EPCs体外迁移及粘附功能的影响及其机制。方法：将PcDNA3.1-Foxc2质粒用脂质体转染EPCs,转染48小时后用定量RT-PCR及Western blot检钡Foxc2基因的表达。用免疫荧光、Western blot、流式细胞仪及定量RT-PCR方法检测CXCR4的表达。用Transwell小室检测EPCs向SDF-1α迁移,用静止粘附实验检测EPCs与纤维连接蛋白的粘附。结果：定量RT-PCR及Western blot检测结果显示,PcDNA3.1-Foxc2质粒转染显著增加Foxc2 mRNA及蛋白的表达。免疫荧光、Western blot及流式细胞仪结果表明Foxc2过表达增加EPCs CXCR4蛋白的表达,定量RT-PCR检测显示Foxc2-EPCs CXCR4 mRNA水平大约是对照组的2倍(P＜0.05)。在SDF-1α刺激下,Foxc2-EPCs迁移及粘附能力明显高于Ctrl-EPCs,而AMD3100或LY294002可抑制Foxc2-EPCs增加的迁移及粘附效应。结论：Foxc2过表达能有效增加EPCs CXCR4的表达及体外迁移、粘附功能,Foxc2过表达所增强的迁移及粘附效应与CXCR4表达上调及下游PI3K/Akt信号活化增强有关。第三部分Foxc2过表达对内皮祖细胞归巢及促内皮修复作用的影响目的：研究Foxc2过表达对EPCs体内归巢及促内皮修复作用的影响。方法：建立小鼠颈动脉内膜损伤模型。从野生小鼠和GFP小鼠骨髓分离培养EPCs和GFP/EPCs,进行Foxc2质粒转染,再经尾静脉输注颈动脉内膜损伤小鼠体内,3天后观察损伤部位GFP标记细胞数目,以检测移植EPCs在体内归巢潜能；7天后用Evens蓝染色检测损伤血管再内皮化的程度,14天后观察GFP+内皮细胞的百分比,以检测移植GFP/EPCs掺入修复内皮层的情况,28天后组织学检测新生内膜和中膜面积的比值(N／M),评价EPCs移植抑制新生内膜增生的治疗效应。结果：GFP/EPCs移植3天后,GFP+严格局限于损伤部位的腔表面,Foxc2-GFP/EPCs移植组损伤血管募集的GFP+细胞明显多于Ctrl-GFP/EPCs组(约为Ctrl-GFP/EPCs组的2倍,P＜0.05)；7天后,Ctrl-EPCs移植组损伤血管再内皮化程度明显提高,Foxc2-EPCs组再内皮化程度又显著高于Ctrl-EPCs组(90.3±1.6%vs 57.2±1.3%,P＜0.05)；14天后,Foxc2-GFP/EPCs移植组GFP+内皮细胞百分比显著高于Ctrl-GFP/EPCs组(46.67±7.09%vs 31.50±5.26%,P＜0.05)；28天后,Ctrl-EPCs移植组小鼠损伤血管新生内膜的形成明显减少,N／M较PBS组降低了65%,而Foxc2-EPCs组新生内膜的增生程度降低更明显(N／M：0.38±0.03 vs 0.67±0.05,P＜0.05)。最后,Foxc2-EPCs移植前用AMD3100或LY294002预孵育能显著抑制Foxc2过表达所增强的EPCs归巢能力、促进再内皮化及抑制新生内膜增生的效应。结论：Foxc2过表达可促进EPCs在损伤内膜的归巢和募集,相应提高EPCs促内皮修复及抑制新生内膜增生的效应；而且Foxc2过表达增强的EPCs归巢及治疗效应与CXCR4/PI3K/Akt信号通路有关。更多还原

【Abstract】 Endothelial disruption or dysfunction is vital to the initiation and progression of atherosclerosis and postangioplasty restenosis. Thus, maintenance of endothelial integrity and promotion of early re-endothelialization are of paramount importance for reducing cardiovascular diseases and the post-intervention complications. Accumulating evidences suggest that endothelial progenitor cells (EPCs) mobilized or transfused are capable of facilitating endothelial repair through direct differentiation into endothelial cells and/or via the paracrine mechanisms. The vasoregenerative effects of EPCs depend on their homing to the vascular injury sites. Indeed, the number of recruited EPCs appears to be related to their circulating numbers but also the functional properties of EPCs homing. Studies show that CXCR4 is a key molecule in regulating EPCs homing and recruitment. Emerging evidences exist for the impaired CXCR4-dependent homing and the low engraftment of EPCs due to some risk factors for coronary artery disease. These underline the need for new strategies capable of increasing the CXCR4-mediated homing potential of EPCs.The Foxc2 protein, a member of the Forkhead/Fox transcription factor family, is essential for the cardiovascular system. Foxc2 plays an important role in the combinatorial regulation of endothelial gene expression and vasculogenesis during embryonic development. Foxc2 is recognized as a novel regulator of angiogenesis via induction of integrinβ3 and angiopoietin-2 expression. Foxc2 might be involved in the angiogenesis under pathologic conditions. Just as recent studies reveal that Foxc2 expression is associated with the human heart failure and increased in periinfarcted zones of the rat left ventricle. Notably, Foxc2 directly induces the expression of CXCR4 in endothelial cells. Recently, it is accepted that the phenotypic and functional behavior of endothelial colony forming cells (ECFCs, also called "late" EPCs) is very similar to mature endothelial cells. Until now there are no data showing the effects of Foxc2 on the functional properties of EPCs. Based on the effects of Foxc2 on the properties of endothelial cells, we sought to determine whether Foxc2 may affect CXCR4 expression and the homing capacity of EPCs as well as the EPCs-mediated endothelial repair.Part One Isolation and Characterization of Mouse Bone Marrow-derived Endothelial Progenitor CellsObjective:To isolate and culture mouse bone marrow-derived EPCs, and to identify the morphological, immunophenotypic and functional characteristics.Methods:Isolated mononuclear cells were cultured in endothelial cell basal medium-2 (EBM-2) supplemented with endothelial growth medium (EGM). After 24 h, nonadherent cells were removed. Adherent cells were cultured for further 15-21 days. Morphological characteristics were observed by inverted microscope. Immunofluorescence, western blot and flow cytometry were used to detect the endothelial cell markers and the hematopoietic markers. The functional characteristics were assessed by uptake of Dil-acLDL and Matrigel tube formation assay.Results:Isolated bone marrow derived-mononuclear cells were cultured for 15-21 days, which exhibited a cobblestone-like morphology. Immunofluorescence and western blot analysis demonstrated that the adherent cells expressed the endothelial cell markers, including CD31, VE-cad, FLK-1 and vWF. Flow cytometric analysis revealed that only very few cells expressed the hematopoietic marker CD34 or leukocytic marker CD45. Moreover, the majority of adherent cells were found positive for uptake of DiI-acLDL. In addition, these cells were capable of assembling into tube-like structures when plated in matrigel. On the basis of the morphological, immunophenotypic and functional characteristics, the cells in our study were confirmed EPCs characterized as ECFCs or late EPCs.Conclusions:Isolated bone marrow derived-mononuclear cells are cultured in EBM-2 supplemented with EGM, which give EPCs.Part Two Effects of Foxc2 Overexpression on Migration and Adhesion Potential of Endothelial Progenitor CellsObjective:To investigate the effects and mechanisms of Foxc2 overexpression on the in vitro migration and adhesion potential of EPCsMethods:EPCs were transfected by using lipidosome transfection reagent with Foxc2 expression vector (Foxc2-EPCs) or with empty control vector (Ctrl-EPCs) and examined 48 hours later. Foxc2 expression of EPCs was detected by western blot and quantitative RT-PCR (qRT-PCR). CXCR4 expression of EPCs was detected by immunofluorescence, western blot, flow cytometry and qRT-PCR. The migration of EPCs toward SDF-la was evaluated in a transwell migration assay, and the adhesion to fibronectin was determined using a static adhesion assay.Results:qRT-PCR and Western blot showed that the transfection with Foxc2 expression vector efficiently induced the up-regulation of Foxc2 mRNA and protein in EPCs. Flow cytometry showed that Foxc2 overexpression significantly increased surface expression of CXCR4 on EPCs. Fluorescence microscopy and western blot analysis further confirmed that Foxc2 overexpression up-regulated CXCR4 expression of EPCs. qRT-PCR analysis showed CXCR4 mRNA expression in Foxc2-EPCs was about 2-fold of Ctrl-EPCs (P<0.05). Foxc2-EPCs demonstrated an increased response to SDF-1α-mediated chemotaxis and adhesion. However, the increased effects were reduced by pretreatment of Foxc2-EPCs with the AMD3100 or LY294002.Conclusions:Foxc2 overexpression may markedly increase CXCR4 expression in EPCs and enhance the in vitro migration and adhesion capacities of EPCs. Moreover, the enhanced in vitro function by Foxc2 overexpression is associated with the up-regulation of CXCR4 and the activation of PI3K/Akt signal pathway.Part Three Effects of Foxc2 Overexpression on Homing and Re-endothelialization Potential of Endothelial Progenitor CellsObjective:To investigate the effects of Foxc2 overexpression on the in vivo homing and the re-endothelialization potential of EPCs.Methods:Mouse carotid injury was established using a 0.014-inch-PTCA flexible guide wire. The mice subjected to carotid injury were randomly assigned into experimental groups. EPCs and GFP/EPCs were isolated and cultured respectively from wild type mice and GFP/mice. EPCs and GFP/EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs or Foxc2-GFP/EPCs) or with empty control vector (Ctrl-EPCs or Ctrl-GFP/EPCs). The mice subjected to carotid injury received PBS, Ctrl-EPCs or Foxc2-EPCs by tail vein injection. To examine the homing capacity, at 3 days after GFP/EPCs delivery, the recruited GFP+ cells to the injury sites were detected by fluorescent microscopy. For assessment of re-endothelialization, animals were perfused with Evans blue dye at 7 days after EPCs delivery. At 14 days, the percent of GFP positive endothelial cells in the cross-section of injured arteries was estimated to observe the incorporation of the delivered EPCs into the regenerative endothelial layer. Neointimal formation was assessed by the morphometric analysis for neointima and media area ratio (N/M) at 28 days after EPCs transfusion. Results:At 3 days after GFP/EPCs delivery, GFP+ cells were strictly restricted to the injury sites. The number of recruited GFP cells was significantly higher in the mice transfused with Foxc2-GFP/EPCs compared with Ctrl-GFP/EPCs (about 2-fold of Ctrl-GFP/EPCs). At 7 days after EPCs delivery, the degree of re-endothelialization was significantly higher in mice transfused with Ctrl-EPCs compared with PBS. Nevertheless, Foxc2-EPCs delivery further increased the degree of re-endothelialization relative to Ctrl-EPCs (90.3±1.6% vs.57.2±1.3%, P<0.05). More GFP cells were incorporated into the CD31 positive endothelial layer at 14 days after Foxc2-GFP/EPCs compared with Ctrl-EPCs delivery (46.7±7.1% vs.31.5±5.3%, P<0.05). At 28 days, Ctrl-EPCs delivery led to a 65% reduction in N/M compared with PBS control (P<0.05). However, the inhibitory effect of Foxc2-EPCs delivery was greater than Ctrl-EPCs (0.38±0.03 vs.0.67±0.05, P<0.05). Finally, preincubation with AMD3100 or LY294002 significantly attenuated the enhanced homing and therapeutic potential of Foxc2-EPCs for promoting re-endothelialization and inhibiting neointimal formation.Conclusions:Foxc2 overexpression may increase EPCs homing and recruitment to the sites of vascular injury, and thereby enhance the therapeutic benefit of EPCs for facilitating re-endothelialization and inhibiting neointimal hyperplasia. Moreover, the enhanced in vivo function by Foxc2 overexpression is associated with the CXCR4/PI3K/Akt signal pathway.更多还原