阻塞性睡眠呼吸暂停患者外周血中内皮祖细胞参与内皮修复机制的研究

发布时间：2018-01-02 23:45

  本文关键词：阻塞性睡眠呼吸暂停患者外周血中内皮祖细胞参与内皮修复机制的研究　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 阻塞性睡眠呼吸暂停 间歇低氧 内皮祖细胞 低氧诱导因子-1? 基质细胞衍生因子-1? 血管内皮细胞生长因子 乙醛脱氢酶

【摘要】：背景与目的:阻塞性睡眠呼吸暂停(OSA)的严重程度与心血管疾病的发生密切相关。OSA模式的间歇低氧(IH)是内皮功能障碍的主要病生理机制。低氧程度越重,间歇低氧频率越高,心血管事件发生率亦越高。内皮祖细胞(EPCs)可维持血管内皮间的动态平衡,参与血管修复。EPC数量的降低可预测未来心血管事件的发生。因此本研究通过招募不同程度的OSA患者并对其进行分组,测定不同程度OSA患者外周血EPC的不同亚族及促血管生成因子的水平,并对外周血中单个核细胞(PBMC)进行体外培养,在此基础上测定EPC的增殖、迁移及血管形成能力,从而进一步探讨OSA患者外周血EPC参与内皮修复的机制。实验方法:连续纳入2013年12月-2014年12月于天津医科大学总医院呼吸科睡眠诊疗中心就诊的90例OSA患者,其中男76名,女14名,年龄40-79岁,同期纳入30名性别、年龄相匹配的健康志愿者(对照组),分别进行多导睡眠监测(PSG),依据睡眠呼吸暂停低通气指数(AHI)分为轻中重度。酶联免疫吸附法(ELISA)测定患者外周血低氧诱导因子-1a(HIF-1a)、基质细胞衍生因子-1a(SDF-1a)、血管内皮生长因子(VEGF)水平;密度梯度离心法提取外周血单个核细胞(PBMC),依据乙醛脱氢酶(ALDH)活性联合CD133、CD34、含激酶域插入片段受体(KDR)相应的EPC表面标志物,应用流式细胞仪测定CD133+KDR+EPC、CD133+CD34+EPC、CD34+KDR+EPC、ALDHlo CD34+KDR+EPC的水平;并对PBMCs进行体外培养,测定EC-CFU的增殖能力;通过Transwell细胞模型观察EPC的迁移能力;Western测定EPC表面膜蛋白的表达水平;与HUVEC共培养测定EPC的成管能力。采用SPSS17.0软件分析包进行统计分析。结果:(1)各组患者年龄、性别间均无显著差异;各组患者体质指数差异明显(F=101.800,P=0.000),其中重度OSA组(33.43±2.03)中度OSA(28.57±0.77)轻度OSA组(25.63±2.97)对照组(23.77±2.73);各组患者AHI差异显著(F=127.700,P=0.000),其中重度OSA组(68.43±28.10)中度OSA组(22.73±4.02)轻度OSA组(11.50±3.10)对照组(1.73±1.41);各组患者最低血氧饱和度差异显著(F=141.300,P=0.000),重度OSA组(0.509±0.168)中度OSA组(0.822±0.150)轻度OSA组(0.874±0.020)对照组(0.922±0.186)。(2)各组患者外周血CD133+KDR+EPC、CD133+CD34+EPC、CD34+KDR+EPC水平差异显著(F=34.340、49.250、84.755,P均=0.000),重度OSA组CD133+KDR+EPC(119.20±45.50)中度OSA组(97.34±16.18)轻度OSA组(76.65±31.91)对照组(51.90±18.85);CD133+CD34+EPC水平重度OSA组(76.65±31.91)中度OSA组(45.19±18.23)轻度OSA组(31.91±8.89)对照组(18.71±8.50);CD34+KDR+EPC水平重度OSA组(239.44±87.23)中度OSA组(123.42±32.73)轻度OSA组(86.09±17.03)对照组(53.29±18.78);各组患者外周血中ALDHlo CD34+KDR+EPC水平差异显著(F=37.383,P=0.000),其中轻度OSA组最高(37.69±11.16),重度OSA组最低(13.01±6.36),中度OSA组(20.45±8.99)对照组(29.52±11.15)。(3)各组血清HIF-1?、SDF-1?、VEGF水平差异明显(F=129.500、69.450、1183.000,P均=0.000),其中HIF-1?、SDF-1?、VEGF水平重度OSA组最高(2.56±0.26,2173±316.5,155.6±12.80),对照组最低(1.30±0.21,1180±313.2,36.79±5.59),中度OSA组(1.87±0.35,1971±275.5,98.00±7.00)轻度OSA组(1.70±0.15,1587±241.7,53.29±6.57)。(4)各组患者外周血EC-CFU数量差异显著(F=48.860,P=0.000),其中轻度OSA组EC-CFU数量最多(73.000?20.020),重度OSA组最少(22.070?9.993),中度OSA组(37.330?17.00)对照组(45.970?18.200)。(5)各组患者外周血EPC迁移的数量差异明显(F=26.670,P=0.000),其中轻度OSA组EPC迁移数量最多(62.100?25.560),重度OSA组最少(16.470?7.785),中度OSA组(34.230?21.640)对照组(49.570?23.790)。(6)各组患者EPC表面CXCR4、VEGFR2蛋白表达灰度值差异明显(F=481.700、195.700,P均=0.000),其中轻度OSA组EPC表面CXCR4、VEGFR2蛋白表达灰度值最高(1.263?0.012,1.562?0.023),重度OSA组最低(0.545?0.038,0.745?0.038),中度OSA组(0.699?0.021,0.987?0.020)对照组(0.988?0.018,0.776±0.059)。(7)各组患者平均血管生成长度差异显著(F=120.100,P=0.000),其中轻度OSA组平均血管生成长度最长(3882.000?477.500),重度OSA组最短(2000.000?551.200),中度OSA组(2559.000?303.700)对照组(3423.000?300.600)。结论:(1)BMI是OSA发病的独立危险因素,可增加OSA的患病率,加重OSA的严重程度。(2)OSA患者程度的加重可导致外周血中促血管活性因子的增加,从而动员大量无效的EPC,而具有修复能力的ALDHlo EPC数量并不增加,尤其重度OSA患者甚至减少,可能与内皮的损伤加重有关,需进一步的相关实验研究。(3)轻度OSA患者外周血EPC动员、增殖、趋化及血管形成能力增强,随着OSA程度的加重,EPC的内皮功能特性削弱,导致内皮损伤与修复间的动态失衡,进一步增加心血管疾病的风险。
[Abstract]:Background and objective: obstructive sleep apnea (OSA) intermittent hypoxia is closely related to the occurrence of.OSA mode and the severity of cardiovascular disease (IH) is the main pathophysiological mechanisms of endothelial dysfunction. The more severe intermittent hypoxia and hypoxia, the higher the frequency, the incidence of cardiovascular events is also higher. Endothelial progenitor cells (EPCs) can maintain the dynamic balance between vascular endothelial, reduce the number of.EPC involved in vascular repair can predict future cardiovascular events. In this study, through the recruitment of different degrees of OSA patients and grouping, serum EPC determination in patients with different degree of OSA in different subgroup and levels of angiogenic factors, and external peripheral blood mononuclear cells (PBMC) were cultured in vitro, determination of EPC based on the proliferation, migration and angiogenesis, in order to further explore the mechanism of peripheral blood EPC in patients with OSA in skin repair. Test method: 90 OSA patients into the December 2013 -2014 year in December in the Department of respiration of General Hospital Affiliated to Tianjin Medical University Sleep Clinic Center Hospital, 76 males, 14 females, age 40-79 years, 30 over the same period included gender, age matched healthy volunteers (control group), respectively by polysomnography (PSG), on the basis of sleep apnea hypopnea index (AHI) were divided into mild and severe. Enzyme linked immunosorbent assay (ELISA) assay in peripheral blood of patients with hypoxia inducible factor -1a (HIF-1a), stromal cell derived factor -1a (SDF-1a), vascular endothelial growth factor (VEGF) level; extraction of peripheral blood mononuclear cells by density gradient centrifugation method (PBMC), on the basis of aldehyde dehydrogenase (ALDH) activity combined with CD133, CD34, containing the kinase domain receptor fragment (KDR) EPC surface markers corresponding to the determination of CD133+KDR, +EPC, flow cytometry was applied to CD133+CD34+EPC, CD34+KDR+EPC, ALDHlo, CD34+KDR+EPC The level of PBMCs; and were cultured in vitro, determination of the proliferation of EC-CFU; to observe the migration ability of EPC cells by Transwell model; Determination of expression of EPC membrane protein Western; co cultured with HUVEC for the determination of EPC tube formation ability. Using SPSS17.0 software analysis package for statistical analysis. Results: (1) age groups there were no significant differences between gender, BMI; patients in each group were significantly different (F=101.800, P=0.000), the severe OSA group (33.43 + 2.03) and moderate OSA (28.57 + 0.77) mild OSA group (25.63 + 2.97) and control group (23.77 + 2.73); each group AHI significantly (F=127.700, P=0.000), which severe OSA group (68.43 + 28.10) in moderate OSA group (22.73 + 4.02) mild OSA group (11.50 + 3.10) and control group (1.73 + 1.41); patients in each group were the lowest oxygen saturation significantly (F=141.300, P=0.000), severe OSA group (0.509 + 0.168) in moderate OSA group (0.822 + 0.150) mild OSA group ( 0.874卤0.020)瀵圭収缁，

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