【作者】 杨兆华；

【导师】 王春生；

【作者基本信息】 复旦大学 ， 外科学， 2009， 博士

【摘要】 在过去的40年中,虽然器官移植术后急性排斥反应(acute rejection,AR)得到了有效的控制,但移植性动脉硬化(transplant arteriosclerosis,TA)却严重制约着移植受者的长期生存。移植性动脉硬化的病理改变特点为移植物内各级动脉内膜发生弥漫性、向心性增厚,引起管腔狭窄。早期病变主要表现为血管壁炎症细胞的浸润,晚期则主要以内膜增厚,管腔狭窄为主。目前认为,移植性动脉硬化是免疫因素和多种非免疫因素共同造成的血管内膜反复损伤、修复的结果。但是,迄今为止,其确切的发病机制仍不清楚,因此,缺乏有效的预防和治疗措施。内皮祖细胞(endothelial progenitor cells,EPCs)作为一类具有多种分化潜能、自我增殖等干细胞性质的特殊细胞,在参与组织损伤、修复及血管形成中发挥重要作用,并与多种心血管疾病的发病密切相关。最近研究表明:EPCs参与移植性动脉硬化发病过程。虽然,移植性动脉硬化的确切发病机制尚未阐明,但是,近几年在EPCs研究领域所取得的诸多成果为探究移植性动脉硬化的发病机制提供了新的思路和依据。大量研究显示,移植性动脉硬化患者外周血EPCs数量明显减少,而且受体源性EPCs可以归巢至移植物血管内膜损伤部位,分化为内皮细胞和/或血管平滑肌细胞并参与移植性动脉硬化内膜增生。但是,受体源性EPCs在移植性动脉硬化发病中的具体作用,尚有争议;EPCs归巢至移植物血管内膜增殖、分化为内皮细胞具体机制,仍有待于做进一步研究。因此,探讨EPCs在移植性动脉硬化的具体作用及相关机制,不仅可望为揭示移植性动脉硬化的发病机理提供重要线索,进而为治疗提供了新的方法。本研究首先通过同种异品系小鼠腹主动脉移植建立移植性动脉硬化模型,并用流式细胞仪检测其外周血EPCs数量变化,探讨EPCs数量动态变化与移植性动脉硬化病变之间的关系。继之,将体外培养扩增的骨髓源性EPCs输入移植性动脉硬化模型体内,观察增加EPCs数量对移植性动脉硬化的影响,探讨受体源性EPCs在移植性动脉硬化中的作用。由于VEGFR-2可在EPCs表面表达并发挥重要作用,因此,在第三部分通过酪氨酸激酶抑制剂Vandetanib特异性阻断VEGFR-2,观察在体外条件下对骨髓单个核细胞向EPCs分化及对后者的生物学功能影响。最后,在小鼠腹主动脉移植术后用Vandetanib进行干预,观察Vandetanib影响EPCs数量及生物学功能情况,从而判断其对移植动脉硬化可能具有的抑制作用。第一部分小鼠移植性动脉硬化模型的建立及外周血EPCs数量动态变化目的:建立小鼠移植性动脉硬化模型,观察移植性动脉硬化病变过程中外周血EPCs数量的动态变化及意义。方法:以C57BL/6小鼠为供体,BALB/c小鼠为受体,建立同种异品系腹主动脉移植模型。光镜和电镜观察移植动脉的病理改变,并采用计算机图像分析系统分析术后2周、4周及6周移植血管内膜增生情况。流式细胞仪检测术前及术后1、3、7、14及28天外周血EPCs数量变化。结果:术后3天,移植动脉内膜损伤并伴有明显的炎症细胞浸润。术后2周,观察到移植动脉内膜增厚,术后4周、6周内膜增生逐渐加重,移植动脉管腔明显狭窄。外周血EPCs数量术后早期增多,3天达到高峰,此后迅速减少,术后2周、4周显著低于术前水平。结论:同种异品系小鼠(C57BL/6→BALB/c)腹主动脉移植成功复制出移植性动脉硬化的病变特点,可用于移植性动脉硬化发病机理和防治措施的研究。外周血EPCs数量的动态变化与移植性动脉硬化病变密切相关,可成为预测移植物动脉硬化发病的新指标。第二部分输入受体骨髓源性EPCs对移植性动脉硬化的促进作用目的:探讨输入体外培养扩增的受体骨髓源性EPCs对移植性动脉硬化的影响。方法:密度梯度离心法分离小鼠骨髓源性单个核细胞,并以贴壁培养法诱导培养EPCs。采用C57BL/6小鼠为供体,BALB/c小鼠为受体,通过同种异品系腹主动脉移植建立小鼠移植性动脉硬化模型。EPCs移植组移植术后通过尾静脉输入EPCs,对照组则注入相应体积的生理盐水。同时将CM-DiI标记的EPCs移植给受体小鼠,进行EPCs移植后体内示踪。术后2、4周处死受体小鼠,观察各组移植动脉的病理改变,用扫描电镜和Evans blue染色方法评价术后14天内皮损伤修复情况;并用计算机图像分析系统测量术后4周移植动脉内膜增生情况。结果:骨髓源性单个核细胞体外可诱导培养出EPCs,表现为其细胞表面既表达干细胞标记(Sca-1),也表达内皮细胞特异性标记(Flk-1,CD31,vWf等),并具有内皮细胞的生物学功能。CM-DiI标记的EPCs移植后主要定位于移植动脉损伤内膜部位。术后2周,EPCs移植组移植动脉内皮损伤较对照组明显加重。术后4周,EPCs移植组移植动脉新生内膜厚度较对照组明显增加,管腔狭窄程度显著加重。结论:EPCs参与移植动脉新生内膜形成,受体骨髓源性EPCs可加重移植动脉内膜损伤,对移植性动脉硬化起促进作用。第三部分Vandetanib对EPCs数量和生物学功能的抑制作用及机制目的:检测VEGFR-2在EPCs中的表达,并观察VEGFR-2抑制剂Vandetanib对骨髓单个核细胞向EPCs分化以及EPCs增殖、黏附和迁移等生物学功能的抑制作用及机制。方法:采用密度梯度离心法从小鼠骨髓获取单个核细胞,将其接种在纤维连接素包被的培养板,加入不同浓度Vandetanib,培养7天后,在倒置荧光显微镜下对EPCs进行计数,观察Vandetanib对骨髓单个核细胞向EPCs分化的影响。收集诱导培养7天的EPCs,按不同Vandetanib干预浓度进行分组,分别用细胞计数、MTT比色法、改良的Boyden小室及黏附能力测定检测Vandetanib对EPCs增殖、迁移和黏附能力的影响。Western blotting法检测EPCs在不同浓度Vandetanib干预后VEGFR-2和pVEGFR-2,Akt和pAkt,Erk和pErk等蛋白表达变化。结果:Vandetanib显著抑制骨髓单个核细胞向EPCs分化。Vandetanib对EPCs的增殖、黏附和迁移能力也有明显抑制作用,并具有浓度和时间依赖性。Westernblotting法检测结果显示EPCs表达VEGFR-2,VEGF可刺激EPCs VEGFR-2的磷酸化。Vandetanib能阻断VEGF诱导的EPCs VEGFR-2的活化,并降低其下游信号分子Akt及Erk磷酸化水平。结论:Vandetanib可阻止骨髓单个核细胞向EPCs分化,并明显抑制EPCs的增殖、黏附及迁移等生物学功能。Vandetanib的这一作用可能与直接阻断VEGFR-2的活化及下游Akt和Erk信号通路有关。第四部分:Vandetanib抑制EPCs对移植性动脉硬化的影响目的:观察VEGFR-2抑制剂Vandetanib对小鼠移植动脉模型外周血EPCs数量的抑制作用及移植动脉新生内膜形成的影响,探讨Vandetanib具有抗移植性动脉硬化作用及可能机制。方法:采用C57BL/6小鼠为供体,BALB/c小鼠为受体,建立同种异品系腹主动脉移植模型。术后将实验动物随机分为3组:①移植对照组;②Vandetanib小剂量干预组(25mg/kg.d);③Vandetanib大剂量干预组(100mg/kg.d)。流式细胞仪分别检测术前及术后1、3、7、14及28天外周血EPCs数量变化;扫描电镜和Evans blue染色方法评价术后14天内皮损伤修复情况;光镜下观察各组移植动脉术后4周的病理改变,并用计算机图像分析系统分析移植动脉新生内膜增生情况。结果:Vandetanib明显降低腹主动脉移植术后早期外周血EPCs数量,术后2周、4周外周血EPCs数量仍维持在较低水平,其中大剂量干预组EPCs数量减少更加显著。术后2周,扫描电镜和Evans blue染色均提示Vandetanib干预组移植动脉内皮损伤较对照组减轻。移植术后4周,Vandetanib大剂量干预组移植动脉内膜增生厚度、炎症细胞浸润程度明显低于对照组(p＜0.05),而Vandetanib小剂量干预组与对照组比较,对内膜增生的抑制作用不明显。结论:大剂量Vandetanib可以明显抑制小鼠移植腹主动脉内膜增生,具有一定的抗移植性动脉硬化的作用。其机制可能与抑制小鼠骨髓单个核细胞向EPCs分化,减少外周血EPCs数量及抑制EPCs生物学功能有关。创新点1.首次发现和报道了小鼠移植性动脉硬化病变过程中外周血EPCs数量的动态变化;2.首次发现和报道了体外培养扩增的EPCs可以加重移植动脉内膜损伤,具有促进移植性动脉硬化作用。3.首次发现和报道了VEGFR-2抑制剂Vandetanib通过减少EPCs数量及抑制其生物学功能,能明显抑制小鼠移植腹主动脉内膜增生,防治移植物血管病变。更多还原

【Abstract】 Over the last 4 decades,although major improvements have been made in the prevention and treatment of acute transplant rejection,transplant arteriosclerosis(TA) in the arteries still limits the long-term survival of patients with solid organ transplantation.TA is characterized by diffuse,uniform,concentric narrowing of the artery owing to proliferative,fibrocellular intima that often results in transplant organ failure.A hallmark of lesions is mononuclear cell infiltration into the vessel wall of grafts at the early stage,followed by neointimal formation.Recent insights have underscored the fact that vascular lesions are the result of cumulative endothelial injuries induced both by alloimmune responses and by nonspecific insults(including ischemia-reperfusion injury,viral infections,and metabolic disorders) in the context of impaired repair mechanisms.However,the precise pathogenetic mechanisms leading to TA are largely unknown and,as a result,adequate prevention and treatment protocols are still lacking.Endothelial progenitor cells(EPCs) can differentiate into a variety of cells to replace dead cells or to repair damaged tissues and seem to be crucial in the development of cardiovascular diseases.Recent evidence indicates that EPCs are involved in the pathogenesis of TA.Although the pathogenesis of transplant arteriosclerosis is not yet fully understood,recent developments in EPCs research have suggested novel mechanisms of vascular remodeling in allografts.For example, EPCs derived from the recipient may repair damaged endothelial cells of arteries in transplant organs and TA is associated with reduction in circulating EPCs.Further evidence suggests that EPCs may be released from both bone marrow and non-bone marrow tissues and appear to replenish cells that died in donor vessels.Concomitantly, EPCs may also accumulate in the intima,where they differentiate into endothelial cells and smooth muscle cells.However,several issues concerning the contribution of EPCs to the pathogenesis of TA are controversial.So,it is highly important to clarify the role of EPCs in transplant arteriosclerosis and understand the detailed mechanisms of EPCs homing and differentiation into mature endothelial cells,and highlights the controversial issues in the field.Based on the understanding to the novel mechanisms of the pathogenesis of TA,we may be able to design a new drug or a new therapeutic strategy to direct EPCs for the prevention of transplant arteriosclerosis.In the present study,we first establish a model of TA in the mouse and investigate whether number of EPCs from peripheral blood correlate with TA severity; then,we intravenous transfusion of ex vivo-expanded bone marrow-derived EPCs to evaluate the role of EPCs in the intimal proliferation in a mouse model of transplant arteriosclerosis;Thereafter,we investigate the affects of vascular endothelial growth factor receptor tyrosine kinase inhibitors vandetanib on the number and functions of EPCs;finally,we investigate the effect of VEGF receptor tyrosine kinase inhibitors Vandetanib on the intimal proliferation in mouse aortic allografts and prospectively construct a new therapeutic strategy for the prevention of TA.PART 1.Mouse Model of Transplant Arteriosclerosis and Changes in the Number of Endothelial Progenitor Cells from peripheral bloodObjective:To establish a model of transplant arteriosclerosis in the mouse and investigate changes in the number of EPCs from peripheral blood.Methods:A segment of abdominal aorta was transplanted orthotopicaUy from C57BL/6 to BALB/c mice.The grafts were harvested at various times after the operation and studied by light and electronic microscopy.Regional changes in the lumen and intimal were measured with computer imaging analysis system.EPCs from peripheral blood were defined by the expression of Sea-1,Flk-1,and quantified by flow cytometry on days 0,1,3,7,14,28 days postoperation.Results:In this study,endothelium injury and inflammatory cells infiltration were seem in the aortic allograft at 3 days after transplantation.Neointimal lesions were observed as early as 2 weeks after surgery and had progressed at 4 and 6 weeks postoperatively.The lumen of allografts were significantly narrowed due to neointima hyperplasia.The number of circulating EPCs increased from 1 days after operation and got to summit at 3 days after the operation.Thereafter EPCs decreased rapidly and were significantly lower at 4 weeks and 6 weeks postoperation than those of pre-operation.Conclusion:Abdominal aortic transplantation from C57BL/6 to BALB/c mice present typical pathological feature of transplant arteriosclerosis.This model may become a simple and powerful tool for studying the pathogenesis and therapeutic intervention for this disease.EPCs count may be considered a novel biological marker of transplant arteriosclerosis.PART 2:Administration of ex vivo-expanded bone marrow-derived EPCs contribute to the development o neointimal thickenings in mouse aortic allograftsObjective:To investigate the effect of bone marrow-derived EPCs on the intimal proliferation in a model of transplant arteriosclerosis in the mouse.Methods:EPCs were isolated from bone marrow-derived mouse mononuclear cells (MNCs) by using a Ficoll density gradient centrifugation and cultured in endothelial basal medium.To establish a model of transplant arteriosclerosis,a segment of abdominal aorta was transplanted orthotopically from C57BL/6 to BALB/c mice. 1×10~7 EPCs or same dosage vehicle(0.9%NS) from the operation day to 2 days post operation were transplanted into the recipients by intravenous transfusion.Meanwhile EPCs were labeled by CM-DiI for cells tracking in vivo.The grafts were harvested at various times after the operation and fluorescence-labeled EPCs,endothelial regeneration rate and regional changes in the lumen and intimal were detected.Results:The adherent cells were considered EPCs that showed spindle shape and had many endothelial characteristics.Two weeks after transplantation,CM-DiI labeled EPCs were detected within the neointima and on the luminal surface of injured aortic allografts.Transplantation of EPCs compared with saline administration markedly accelerated endothelium injury of allografts.Four weeks postoperation,the neointimal thickness in transplantation group was significantly higher than that of control group.Conclusion:EPCs may participate in the formation of the vascular lesion in aortic allografts.Bone marrow-derived EPCs promote endothelial damage and contribute to the neointima formation in a murine model of transplant arteriosclerosis.PART 3:Effect and mechanism of vascular endothelial growth factor receptor tyrosine kinase inhibitors vandetanib on the number and functions of EPCsObjective:To investigate expression of Vascular endothelial growth factor receptor 2(VEGFR-2) on EPCs and effects of VEGF receptor tyrosine kinase inhibitors Vandetanib on the number and proliferation,adhesion,migration functions of EPCs and possible mechanisms associated with Akt and Erk signaling pathway.Methods:To study the effect of vandetanib on MNCs differentiation into EPCs, mononuclear cells(MNCs) were isolated from mouse bone marrow by using a Ficoll density gradient centrifugation and cultured on fibromectin-coated culture dishes with or without vandetanib for 7 days.EPCs were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under fluorescence microscopy. After 7 days cultured,attached cells were treated with vandetanib(0.25～4uM) or vehicle for various time points.EPCs proliferation,migration were assayed with MTT assay and modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating them on fibronectin-coated dishes,and then adherent cells were counted.Western blotting was used to analysis the effect of vandetanib on VEGFR-2 activation and Akt and Erk phosphorylation of EPCs.Results:EPCs number differentiated from MNCs at 7 days was significantly lower in vandetanib treated cells in a dose-dependent manner than that of vehicle-treated cells.Vandetanib also significantly inhibited the proliferative,migratory and adhesive capacity of EPCs in a time and dose dependent manner.Western blotting analysis showed that VEGFR-2 were activated in EPCs after sitmulated with VEGF. Vandetanib potently inhibited phosphorylation of VEGFR-2 in a dose-dependent manner.VEGFR-2 blockade corresponded with decreased phosphorylation of downstream signaling intermediates Akt and Erk.Conclusion:Present results suggested that vandetanib could inhibit EPCs differentiation from MNCs and reduce the proliferation,migration and adhesion capacities of EPCs,possibly because it suppress Akt and Erk pathways activation by blocking the activation of VEGFR-2.PART 4:Potent inhibitory effect of Vandetanib on endothelial progenitor cells prevents Transplant Arteriosclerosis in mouse aortic allograftsObjective:To investigate the effect of VEGF receptor tyrosine kinase inhibitors Vandetanib on the number of endothelial progenitor cells(EPCs) from peripheral blood and the intimal proliferation in mouse aortic allografts and the possible mechanism on preventing transplant arteriosclerosis.Methods:To establish a model of transplant arteriosclerosis,a segment of abdominal aorta was transplanted orthotopicaUy from C57BL/6 to BALB/c mice.The recipients were divided into three groups:the allografts control group,low-dose vandetanib treated group(25mg/kg.d) and high-dose vandetanib treated group(100mg/kg.d). EPCs from peripheral blood were defined by the expression of Sea-1,Flk-1,and quantified by flow cytometry on days 0,1,3,7,14,28 days postoperation.At 2 weeks after surgery,scanning electron microscope and Evans blue staining were used to measure the reendothelialized area of aortic allografts.Animals were sacrificed and the aortic grafts were harvested at 4 weeks post operation.The aortic grafts were then processed for staining and measurements by means of pathology.Regional changes in the lumen,intimal and medial layers were measured with computer imaging analysis system.Results:The number of circulating EPCs were significantly reduced in group treated with vandetanib compared with those of control group early after the operation.At 2 weeks and 4 weeks postoperation,the level of circulating EPCs in vandetanib treated group still lower than those of pre-operation,especially in high-dose vandetanib treated group.At 2 weeks after surgery,the rate of reendothelialization in vandetanib treated group was much higher than that of control group.At 4 weeks post operation, intimal thickening was significantly reduced and inflammatory cells were suppressed in aortic allografts in high-dose vandetanib treated group,but not in low-dose vandetanib treated group.Conclusion:High-dose vandetanib can inhibit intimal proliferation in aortic allografts and prevent the transplant arteriosclerosis.The mechanisms are related to inhibit the differentiation of EPCs from the bone marrow,reduce the number and suppress function of circulating EPCs.更多还原