【作者】 刘哲亮；

【导师】 胡建国；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 背景与目的：内皮祖细胞(Endothelial Progenitor Cells, EPCs)的数量和功能与血管内皮功能明确相关。细胞复制性衰老是指细胞失去分化能力,同时发生相应形态学改变和其它功能障碍。危险因素通过氧化机制加速EPCs的衰老进程。残粒脂蛋白(Remnant lipoproteins, RLPs)可通过氧化机制直接导致血管内皮舒张功能损害和成熟内皮细胞分泌功能的异常,但是还不清楚RLPs是否还影响EPCs衰老及其功能。本研究拟观察RLPs对EPCs衰老、增殖、迁移、粘附和体外血管新生能力的影响。方法：采用已建立的高脂餐方案：总热量为800千卡(脂肪50克、蛋白质28克、碳水化合物60克),采集高甘油三酯血症患者脂餐后4小时EDTA抗凝静脉血标本。应用免疫亲和层析法和密度梯度超速离心技术分离人血浆RLPs, BCA法测定RLPs的蛋白含量。密度梯度离心法分离人外周血的单个核细胞,利用添加生长因子的M 199条件培养基诱导单个核细胞贴壁向EPCs分化。流式细胞仪、双荧光染色、直接免疫荧光染色和普通免疫细胞化学染色鉴定EPCs。1)将不同浓度(0-200μg/mL)的RLPs加入第10天的EPCs,分别培养6、12、24及48小时。采用衰老相关的p-半乳糖苷酶(senescence-associatedβ-galactosidase, SA-β-Gal)染色技术,在PH6的条件下,计数强SA-β-gal活性的EPCs,获取RLPs影响EPCs衰老进程的最大效应浓度(CR)和最佳干预时间(TR)。2)将培养第10天EPCs以添加CR浓度RLPs的M199条件培养液培养TR时间,再加入不同浓度(0.01-10μmol/L)的阿托伐他汀(Atorvastatin,ATR),分别培养6、12、24及48小时。采用SA-β-gal染色技术,在PH6的条件下,计数强SA-β-gal活性的EPCs,获取ATR影响EPCs衰老进程的最大效应浓度(CA)和最佳干预时间(TA)。3)分为空白对照组、RLPs刺激组、ATR干预组和RLPs刺激+ATR干预组。将浓度为CR的RLPs、浓度为CA的ATR和对照物于细胞培养第10天加入EPCs,分别作用相应时间。采用SA-β-gal染色技术,在PH6的条件下,计数强SA-β-gal活性的EPCs,检测各干预措施对EPCs衰老的影响。4)观察RLPs对EPCs功能的影响分为空白对照组、RLPs刺激组、ATR干预组和RLPs刺激+ATR干预组。将浓度为CR的RLPs、浓度为CA的ATR和对照物于细胞培养第10天加入EPCs,分别作用相应时间。①改良的Boyden小室评价细胞迁移能力。②粘附能力测定实验评价细胞粘附能力。③MTT比色法评价细胞增殖能力。④Matrigel成血管网实验评价EPCs在体外的血管新生能力。结果：(1)外周血单个核细胞用添加生长因子的M199条件培养基培养可诱导分化成EPCs,具备典型的形态学和表型特征；(2)从高甘油三酯血症患者静脉血中制得RLPs,经BCA法测定的RLPs蛋白浓度值为4.28±0.36mg/mL;(3) RLPs的最大效应浓度为100μg/mL,最佳干预时间为24小时；ATR的最大效应浓度为1.0μmol/L,最佳干预时间为24小时；(4) RLPs刺激能使EPCs的SA-β-Gal活性增加,EPCs衰老细胞数增加,其迁移、粘附、增殖和形成微血管网的能力显著降低；(5)ATR治疗可以降低EPCs的SA-β-Gal活性,显著减少EPCs衰老细胞数,增强其迁移、粘附、增殖和形成微血管网的能力。结论：RLPs可以导致EPCs衰老并抑制其血管新生的相关功能,ATR治疗可以改善RLPs对EPCs的上述负面影响。背景与目的：裸鼠后肢缺血模型是目前国际上最常采用的用于评价EPCs移植治疗效果的实验动物模型。采用裸鼠后肢缺血模型,通过移植DAPI标记的EPCs,并运用免疫组化技术、细胞衰老生物标记染色技术和激光多普勒血流成像技术,观察RLPs对EPCs移植后裸鼠缺血后肢血运重建的影响。方法：参照Heeschen等的方法(Circulation,2004;109:1615-22),应用8-10周龄BALB/c雄性裸鼠建立后肢缺血裸鼠模型,缺血手术24小时后,以PBS液作对照,由尾静脉注入体外扩增培养10天且经DAPI荧光标记的EPCs至裸鼠体内。于EPCs移植第1天开始,每间隔48小时经由尾静脉注入RLPs或对照物；同时根据ATR应用情况,将裸鼠分为：对照组、RLPs刺激组、ATR干预组、RLPs刺激+ATR干预组。激光多普勒血流扫描仪监测后肢的血液灌流情况。21天后,处死裸鼠,取缺血后肢与正常后肢腓肠肌做病理切片。免疫组化法检测缺血组织的新生微血管密度和EPCs的组织分布情况。采用SA-β-gal染色技术,在PH6的条件下,计数强SA-β-gal活性的血管数目。结果：(1)成功建立裸鼠后肢缺血模型,缺血后肢末梢循环、运动功能、血流灌注情况随时间发生规律改变,组织病理学改变符合缺血特征；(2) EPCs移植可以显著降低缺血后肢的伤残率；(3) RLPs刺激组的EPCs组织分布数、新生毛细血管密度和缺血后肢血流灌注要显著低于其他各组,可以观察到新生血管的衰老现象；(4)ATR治疗组的EPCs组织分布数、新生毛细血管密度和缺血后肢血流灌注要显著高于其他各组。结论：RLPs通过刺激EPCs衰老而抑制其在体内的血管新生能力,ATR治疗可以改善EPCs受损的血管新生能力。背景与目的：微小RNA(microRNA, miRNA)是新近证明的一类高度保守的、内源性非蛋白编码的长度约为20-24个核苷酸的小分子RNA,参与调节血管内皮细胞或胚胎干细胞的血管新生,参与调控细胞衰老。通过对RLPs干预体外培养EPCs的研究,利用miRNA芯片技术检测相关miRNA的改变情况,以初步了解miRNA是否参与调控RLPs对EPCs血管新生的影响。方法：取培养第10天EPCs,分为RLPs干预组和对照组,采用Agilent的人microRNA寡核苷酸基因芯片寻找显著差异表达的miRNA,构建RLPs干预组和对照组的miRNA表达谱：用mirVana RNA Isolation Kit提取两组EPCs的总RNA并进行纯化；使用Nanodrop分光光度计(NP-1000)测定RNA在260nm和280nm波长的吸收值,计算样本总RNA浓度并评估纯度；采用Agilent RNA 6000 Nano Assay检测RNA纯度及完整性；采用Agilent’s miRNA Complete Labeling and Hyb Kit进行：miRNA荧光标记和芯片杂交；采用Agilent’s Gene Expression Wash Buffer Kit进行杂交芯片的洗脱；使用Agilent Microarray Scanner扫描芯片的荧光强度并借助Agilent Feature Extraction Software提取原始数据进行分析运算,筛选两组间差异表达显著的miRNA。结果：与对照组相比,EPCs经RLPs干预后显著异常表达的miRNA共有4个,显著上调表达miRNA有3个,分别为：hsa-miR-148b*、hsa-miR-155*和hsa-miR-513c;一个显著下调的miRNA,即hsa-miR-574-3p。结论：miRNA可能参与调控RLPs对EPCs血管新生的影响。更多还原

【Abstract】 Background and Objective:Remnant lipoproteins (RLPs) are closely associated with coronary heart disease and can induce endothelial dysfunction through oxidative mechanisms. Many risk factors accelerate the onset of endothelial progenitor cells (EPCs) senescence via increased oxidative stress. In this study, we investigated the effect of RLPs on EPCs senescence and function.Methods:RLPs were isolated from postprandial plasma of hypertriglyceridemic patients by use of the immunoaffinity gel mixture of anti-apoA-1 and anti-apoB-100 monoclonal antibodies. The mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolated cells were suspended in Medium 199 supplement with 20% fetal blood serum and bovine pituitary extract for culturing, differentiating and proliferating. The expressions of specific antigens on cell surface, such as CD133、KDR、CD34、CD31 and vWF were analyzed by immunocytochemistry on day 10. To assess the onset of senescence, SA-β-gal activity was measured. The numbers of SA-β-gal positive cells were counted manually in each sample under bright field illumination. Modified Boyden chamber assay and MTT assay was used to measure the migration and proliferation function of EPCs.The adhesion assay was performed by replating on fibronectin-coated dishes, then adherent cells were counted. Matrigel Network Formation Assay was performed by replating on Matrigel-coated dishes, then the number of closed loops formed by capillary tubes network was counted.Results:peripheral blood mononuclear cells cultivated with M199 supplement with growth factors generated EPCs as expected, which were characterized by typical morphology and phenotype. EPCs became senescent as determined by senescence-associated acidicβ-galactosidase (SA-β-Gal) staining after ex vivo cultivation without any stimulation. Co-incubation with RLPs accelerated the increase in SA-β-Gal-positive EPCs. The acceleration of RLPs-induced EPCs senescence occurred dose-dependently with a maximal effect when EPCs were treated with RLPs at 100μg/mL (P<0.01). RLPs decreased adhesion, migration and proliferation capacities of EPCs as assessed by adherence to fibronectin, modified Boyden chamber technique and MTT assay (P<0.01), respectively. Moreover RLPs suppressed capillary-like tube formation on Matrigel for all cells tested. However, RLPs-induced EPCs senescence and dysfunction were significantly inhibited by treatment of atorvastatin (ATR,1.0μmol/L)(P<0.01).Conclusions:RLPs accelerate the onset of EPCs senescence via increased oxidative stress, accompanying with the impaired adhesion, migration, proliferation and suppressed capillary-like tube formation capacity. ATR prevents RLPs-induced senescence and EPCs dysfunction. Background and Objective:Hindlimb ischemia model is the widely used animal model, for it involved in the study of angiosclerosis and embolism in lower limb. Recentely, it becames an original tendency that encouraging the neovascularization by EPCs transplantation in animal hindlimb ischemia model. In this study, we investigated the effects of RLPs on therapeutic angiogenesis of circulating endothelial progenitor cells transplanted in hindlimb-ischemic nude mouse.Methods:8-10 weeks old BALB/c male nude mice were operated according to Heeschen’s methods(Circulation,2004; 109:1615-22) to obtain hind limb ischemia model.One day after surgery, 1×106 of DAPI labeled EPCs in 200μL of M199 medium were injected into the caudal vein of mice. A laser Doppler perfusion imager (Moor Instruments), which maps tissue blood flow by the shift in the laser light frequency, was used for serial noninvasive physiological evaluation of neovascularization. Each mouse was followed by serial recording of surface blood flow of hind limb on days 0,3,7,14, and 21. Tissue capillary density was determined as a histological evaluation of neovascularization. To assess the onset of senescence, SA-β-gal activity was measured. The numbers of SA-β-gal positive cells and capillaries were counted manually in each sample under bright field illumination.Results::We establish nude mouse hindlimb ischemia mode with obvious ischemia in mouse’s hindlimb. EPCs transplantation significantly reduce foot necrosis and auto-amputation. Administration of EPCs treated with ATR resulted in a significantly lower rate of limb loss (necrosis of tissue above knee or autoamputation) compared with EPCs treated with RLPs and with control (P<0.01). Serial analysis of laser Doppler perfusion imaging revealed a greater increase of limb perfusion in the ischemic limb of mice injected with EPCs that were treated with ATR than with RLPs(P<0.01). Administration of EPCs treated with ATR increased capillary density of ischemic skeletal muscle compared with EPCs that were treated with RLPs(P<0.01).Conclusions:RLPs accelerate the onset of EPCs senescence and reduce therapeutic angiogenesis of circulating EPCs transplanted in hindlimb-ischemic nude mouse. ATR improves the angiogenesis ability of these EPCs. Background and Objective:MicroRNAs (miRNAs) are small (18-24nt) non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis, proliferation and neovascularization. We investigated the possible mechanism of effects of remnant lipoproteins on angiogenesis of circulating endothelial progenitor cells using an oligonucleotide microchip for microRNA profiling.Methods:After 10 days of culture, EPCs were treated with/ without RLPs,the later were served as control. An oligonucleotide microchip was used to find miRNAs that were differential expressed. Total RNA was extracted using mirVana RNA Isolation Kit for miRNA microarray. Assess the purity of the RNA adopting Agilent RNA 6000 Nano Assay. Between two groups of the fingerprints, Agilent Microarray Scanner and Agilent Feature Extraction Softwarewas performed to screen the markedly differentially expressed genes.Results:Four miRNAs differentially expressed between these two groups were detected. Three are upregulated:hsa-miR-148b*、hsa-miR-155、and hsa-miR-513c, One is downregulated, that is hsa-miR-574-3p (RLPs treated vs. control). Conclusions:MiRNAs may regulate the effect of remnant lipop-roteins on angiogenesis of circulating endothelial progenitor cells.更多还原