【作者】 王仁；

【导师】 姜志胜； 王佐；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 周围血管疾病一直严重危害着人类健康，目前尚无非常有效的治疗方法。内皮祖细胞(endothelial progenitor cells, EPCs)的发现开辟了一条新的治疗途径。EPCs是成熟血管内皮细胞的前体细胞，既可不断增殖，又能定向分化为成熟的内皮细胞，在血管生成中具有多种功能。载脂蛋白(a)[(apolipoprotein(a)，apo(a)]是脂蛋白(a)[lipoprotein(a)，Lp(a)]重要组成成分，也是周围血管疾病独立危险因子。有文献报道了Lp(a)对EPCs生物学功能的影响，但有关Lp(a)对内皮细胞及EPCs的增殖、分化作用，仍存在两种截然对立的实验证据。血清Lp(a)水平不受环境、饮食和药物的影响，而apo(a)的合成情况可影响着Lp(a)血清浓度，并且apo(a)还可独立于apoB-100或Lp(a)而单独影响血管病理生理功能。但apo(a)影响EPCs血管生成及其机制尚不清楚。本研究从小鼠骨髓分离培养EPCs，观察apo(a)对EPCs体内外血管生成能力的影响，并探讨了其机制。主要研究结果及方法如下：第一部分Apo(a)对小鼠骨髓源性EPCs血管生成能力的影响目的：研究apo(a)对小鼠骨髓源性EPCs迁移、粘附、归巢及血管生成能力的影响。方法：从小鼠骨髓分离、培养、鉴定EPCs，观察apo(a)对EPCs粘附、迁移及matrigel胶上血管腔样结构的影响；复制小鼠下肢缺血模型，3×10~5EPCs移植下肢缺血实验小鼠，于移植后第3、7、14天取小鼠腓肠肌，检测缺血组织EPCs归巢数量及毛细血管密度；RT-PCR、western blot检测P/E selectin、PGSL-1、CXCR4、VEGF表达。结果：小鼠骨髓源性EPCs可吞噬Dil-AcLDL并结合UEA-1，原代EPCs细胞表面标志CD133+/VEGFR-2+、CD34+/VEGFR-2+、vWF分别为72.2%、71.26%和1.0%。Apo(a)呈剂量依赖性抑制EPCs粘附能力：加入0.2μg/ml apo(a),与对照组相比，粘附率减少94.2%(P<0.01)，加入15μg/ml apo(a), EPCs几乎完全凋亡，体外迁移实验也取得了相似结果。Mtrigel胶上，10μg/ml apo(a)时，野生型鼠EPCs及转基因鼠EPCs血管腔样结构被破坏92.1%(P<0.01)。RT-PCR及western blot检测到缺血组织血管内皮表达P/E selectin，VEGF在缺血组织表达升高，而PGSL-1、CXCR4在apo(a)处理的EPCs表达降低；动物实验中，对照鼠缺血组织血管周围可见EPCs募集，而转apo(a)基因鼠不但EPCs归巢数量显著减少(P<0.05)，毛细血管生成数量也显著减少(P<0.05)。结论：apo(a)可抑制EPCs粘附、迁移、归巢及血管生成能力。第二部分Apo(a)影响EPCs血管生成能力的机制研究目的：探讨apo(a)抑制EPCs血管生成的机制。方法：收集培养的EPCs，提取总RNA, RT-PCR检测Notch受体、配基及LOX-1表达；分析apo(a)影响它们表达的最大时效、量效关系；免疫共沉淀检测apo(a)对Notch受体、配基结合的影响，RT-PCR检测下游基因表达。结果：小鼠骨髓源性EPCs只检测到Notch2受体和JAG-1、JAG-2配基表达，而且在10μg/ml apo(a)处理24小时表达最高；apo(a)促进Notch2与JAG-1配基结合，RBPJ、HES、HEY的mRNA表达量分别增加30%、27%及22%(P<0.05)，CXCR4、PGSL-1蛋白表达量分别降低72%与80%(P<0.05)，但VEGF表达无显著变化。封闭LOX-1受体后，免疫共沉淀检测不到Notch2受体与JAG-1配基结合，CXCR4、PGSL-1、VEGF表达与LOX-1封闭前相比，无显著差异。结论：apo(a)促进Notch受体配基结合,上调RBPJ、HES、HEY表达，降低CXCR4、PGSL-1表达，抑制EPCs的粘附、迁移、归巢及血管生成能力；LOX-1参与了apo(a)对EPCs血管生成的影响。更多还原

【Abstract】 At present, there are no very effective therapeutic measures for the treatment ofischemic peripheral vascular diseases which has a serious harming to human health.The discovery of endothelial progenitor cells (EPCs) opens up the possibility ofdeveloping angiogenesis therapies because EPCs are precursor cells of endothelialcell, can proliferate, and differentiate into mature endothelial cells. Apolipoprotein(a)[(apo(a)], an important component of lipoprotein (a)[Lp(a)], has been considered tobe an independent risk factor for peripheral vascular disease. Some studies havereported the effect of Lp(a) on EPCs biological functions, but we can not determinewhether Lp(a) impact EPCs proliferation and differentiation because of the twoopposite experimental evidences. Lp(a) level is not influenced by the environment,diet and medication, and its plasma concentration is mainly controlled by the rate ofapo (a) de novo biosynthesis. Furthermore, apo(a) can effect vascular pathological andphysiological function independent of the apoB-100or Lp(a). But the exactmechanism of apo(a) affecting EPCs angiogenesis is unclear.In the present study, we has isolated mouse bone marrow-derived EPCs,investigated the effect of apo(a) on EPCs angiogenesis in vitro and in vivo andexplored its mechanism. The main research results and methods are as follows:Part1Apo (a) affects angiogenesis of mouse bone marrow-derived EPCsObjective: To study the effect of apo(a) on mouse bone marrow-derived EPCs migration, adhesion, homing and angiogenesis abilities.Methods: EPCs were isolated from the bone marrow of apo(a) transgenic mice andwild-type litter mates. These cells were cultured with or without apo(a) beforetransplantation. Hindlimb ischemia models were surgically induced in mice, whichthen received an intravenously injection of3×10~5EPCs. At3,7and14days post EPCtransplantation，the adhesion, migration abilities and capillary density in calf muscleswere assessed. Then, EPCs and muscle tissues were harvested and the expression ofP/E selectin、PGSL-1、CXCR4、VEGF, etc. was detected by RT-PCR and westernblot.Results: Results showed EPCs could take up Dil-AcLDL and bind to FITC UEA-1.The number of EPCs expressing CD133+/VEGFR-2+,CD133+/CD34+and vWF was73.43%,72.12%and1.0%, respectively. Apo(a) inhibited EPCs adhesion ability in adose dependent manner. Addition of0.2μg/ml apo(a), inhibition of94.2%comparedwith control group was observed (P<0.01, n=3); at a concentration of15μg/ml apo(a), EPCs almost completely expressed apoptosis. An in vitro migration experimentalso produced the similar results. After7days cultured, tubule-like formation onmatrigel gels was impaired92.1%treated with10μg/ml apo(a) and almostcompletely eliminated at the concentration of15μg/ml(P<0.01, n=3); RT-PCRexamination disclosed a high expression of P/E-selectin in the endothelium ofischemic tissues compared with normal hindlimb tissues (P<0.05, n=3) and a downregulated expression of PGSL-1on apo (a)-treated EPCs detected by western blot (P<0.05, n=3). Histological examination revealed reduced local accumulation of EPCsand a decreased number of capillary ECs in apo (a) transgenic mice compared withcontrol group (P<0.05, n=8).Conclusion: Apo (a) impaired the abilities of EPCs adhesion, migration and homing,and resulted in an inhibition of EPCs angiogenesis. Part2The study on the mechanisms of apo(a) effecting EPCsangiogenesisObjective: To explore the mechanisms of apo(a) inhibits EPCs angiogenesis.Methods: EPCs were harvested to extract total RNA, then the expression of Notchreceptors, ligands and LOX-1receptors and the greatest time-effect, dose-effectrelationships of apo(a) on them were detected by RT-PCR; Immunoprecipitationmethod was applied to analyze the effect of apo(a) on the receptor-ligands binding,RT-PCR to analyze the expression of downstream genes.Results: Results indicated that only Notch2receptor, JAG-1and JAG-2ligandswere observed on EPCs, all of them had the highest expression after EPCs weretreated by10μg/ml apo(a) for24h; apo(a) could promote the binding ofNotch2-JAG-2,up-regulate the expression of RBPJ、HES、HEY genes (P<0.05,n=3)and down-regulate the expression of CXCR4、PGSL-1(P<0.05, n=3), but therewas no change in VEGF expression. The binding of Notch2-JAG-1was undetectableby immunoprecipitation and the expression of CXCR4、PGSL-1、VEGF had nosignificant differences after LOX-1receptor was blocked.Conclusion: Apo (a) could promote Notch2-JAG-1binding, up-regulate theexpression of RBPJ、 HES、 HEY genes and down-regulate the expression ofCXCR4、PGSL-1, inhibit EPCs adhesion,migration,homing and angiogenesisabilities. LOX-1was involved in the effect of apo (a) on EPCs angiogenesis.更多还原