本文關(guān)鍵詞：阻塞性睡眠呼吸暫停患者外周血中內(nèi)皮祖細(xì)胞參與內(nèi)皮修復(fù)機(jī)制的研究　出處：《天津醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 阻塞性睡眠呼吸暫停 間歇低氧 內(nèi)皮祖細(xì)胞 低氧誘導(dǎo)因子-1? 基質(zhì)細(xì)胞衍生因子-1? 血管內(nèi)皮細(xì)胞生長因子 乙醛脫氫酶

【摘要】：背景與目的:阻塞性睡眠呼吸暫停(OSA)的嚴(yán)重程度與心血管疾病的發(fā)生密切相關(guān)。OSA模式的間歇低氧(IH)是內(nèi)皮功能障礙的主要病生理機(jī)制。低氧程度越重,間歇低氧頻率越高,心血管事件發(fā)生率亦越高。內(nèi)皮祖細(xì)胞(EPCs)可維持血管內(nèi)皮間的動(dòng)態(tài)平衡,參與血管修復(fù)。EPC數(shù)量的降低可預(yù)測未來心血管事件的發(fā)生。因此本研究通過招募不同程度的OSA患者并對其進(jìn)行分組,測定不同程度OSA患者外周血EPC的不同亞族及促血管生成因子的水平,并對外周血中單個(gè)核細(xì)胞(PBMC)進(jìn)行體外培養(yǎng),在此基礎(chǔ)上測定EPC的增殖、遷移及血管形成能力,從而進(jìn)一步探討OSA患者外周血EPC參與內(nèi)皮修復(fù)的機(jī)制。實(shí)驗(yàn)方法:連續(xù)納入2013年12月-2014年12月于天津醫(yī)科大學(xué)總醫(yī)院呼吸科睡眠診療中心就診的90例OSA患者,其中男76名,女14名,年齡40-79歲,同期納入30名性別、年齡相匹配的健康志愿者(對照組),分別進(jìn)行多導(dǎo)睡眠監(jiān)測(PSG),依據(jù)睡眠呼吸暫停低通氣指數(shù)(AHI)分為輕中重度。酶聯(lián)免疫吸附法(ELISA)測定患者外周血低氧誘導(dǎo)因子-1a(HIF-1a)、基質(zhì)細(xì)胞衍生因子-1a(SDF-1a)、血管內(nèi)皮生長因子(VEGF)水平;密度梯度離心法提取外周血單個(gè)核細(xì)胞(PBMC),依據(jù)乙醛脫氫酶(ALDH)活性聯(lián)合CD133、CD34、含激酶域插入片段受體(KDR)相應(yīng)的EPC表面標(biāo)志物,應(yīng)用流式細(xì)胞儀測定CD133+KDR+EPC、CD133+CD34+EPC、CD34+KDR+EPC、ALDHlo CD34+KDR+EPC的水平;并對PBMCs進(jìn)行體外培養(yǎng),測定EC-CFU的增殖能力;通過Transwell細(xì)胞模型觀察EPC的遷移能力;Western測定EPC表面膜蛋白的表達(dá)水平;與HUVEC共培養(yǎng)測定EPC的成管能力。采用SPSS17.0軟件分析包進(jìn)行統(tǒng)計(jì)分析。結(jié)果:(1)各組患者年齡、性別間均無顯著差異;各組患者體質(zhì)指數(shù)差異明顯(F=101.800,P=0.000),其中重度OSA組(33.43±2.03)中度OSA(28.57±0.77)輕度OSA組(25.63±2.97)對照組(23.77±2.73);各組患者AHI差異顯著(F=127.700,P=0.000),其中重度OSA組(68.43±28.10)中度OSA組(22.73±4.02)輕度OSA組(11.50±3.10)對照組(1.73±1.41);各組患者最低血氧飽和度差異顯著(F=141.300,P=0.000),重度OSA組(0.509±0.168)中度OSA組(0.822±0.150)輕度OSA組(0.874±0.020)對照組(0.922±0.186)。(2)各組患者外周血CD133+KDR+EPC、CD133+CD34+EPC、CD34+KDR+EPC水平差異顯著(F=34.340、49.250、84.755,P均=0.000),重度OSA組CD133+KDR+EPC(119.20±45.50)中度OSA組(97.34±16.18)輕度OSA組(76.65±31.91)對照組(51.90±18.85);CD133+CD34+EPC水平重度OSA組(76.65±31.91)中度OSA組(45.19±18.23)輕度OSA組(31.91±8.89)對照組(18.71±8.50);CD34+KDR+EPC水平重度OSA組(239.44±87.23)中度OSA組(123.42±32.73)輕度OSA組(86.09±17.03)對照組(53.29±18.78);各組患者外周血中ALDHlo CD34+KDR+EPC水平差異顯著(F=37.383,P=0.000),其中輕度OSA組最高(37.69±11.16),重度OSA組最低(13.01±6.36),中度OSA組(20.45±8.99)對照組(29.52±11.15)。(3)各組血清HIF-1?、SDF-1?、VEGF水平差異明顯(F=129.500、69.450、1183.000,P均=0.000),其中HIF-1?、SDF-1?、VEGF水平重度OSA組最高(2.56±0.26,2173±316.5,155.6±12.80),對照組最低(1.30±0.21,1180±313.2,36.79±5.59),中度OSA組(1.87±0.35,1971±275.5,98.00±7.00)輕度OSA組(1.70±0.15,1587±241.7,53.29±6.57)。(4)各組患者外周血EC-CFU數(shù)量差異顯著(F=48.860,P=0.000),其中輕度OSA組EC-CFU數(shù)量最多(73.000?20.020),重度OSA組最少(22.070?9.993),中度OSA組(37.330?17.00)對照組(45.970?18.200)。(5)各組患者外周血EPC遷移的數(shù)量差異明顯(F=26.670,P=0.000),其中輕度OSA組EPC遷移數(shù)量最多(62.100?25.560),重度OSA組最少(16.470?7.785),中度OSA組(34.230?21.640)對照組(49.570?23.790)。(6)各組患者EPC表面CXCR4、VEGFR2蛋白表達(dá)灰度值差異明顯(F=481.700、195.700,P均=0.000),其中輕度OSA組EPC表面CXCR4、VEGFR2蛋白表達(dá)灰度值最高(1.263?0.012,1.562?0.023),重度OSA組最低(0.545?0.038,0.745?0.038),中度OSA組(0.699?0.021,0.987?0.020)對照組(0.988?0.018,0.776±0.059)。(7)各組患者平均血管生成長度差異顯著(F=120.100,P=0.000),其中輕度OSA組平均血管生成長度最長(3882.000?477.500),重度OSA組最短(2000.000?551.200),中度OSA組(2559.000?303.700)對照組(3423.000?300.600)。結(jié)論:(1)BMI是OSA發(fā)病的獨(dú)立危險(xiǎn)因素,可增加OSA的患病率,加重OSA的嚴(yán)重程度。(2)OSA患者程度的加重可導(dǎo)致外周血中促血管活性因子的增加,從而動(dòng)員大量無效的EPC,而具有修復(fù)能力的ALDHlo EPC數(shù)量并不增加,尤其重度OSA患者甚至減少,可能與內(nèi)皮的損傷加重有關(guān),需進(jìn)一步的相關(guān)實(shí)驗(yàn)研究。(3)輕度OSA患者外周血EPC動(dòng)員、增殖、趨化及血管形成能力增強(qiáng),隨著OSA程度的加重,EPC的內(nèi)皮功能特性削弱,導(dǎo)致內(nèi)皮損傷與修復(fù)間的動(dòng)態(tài)失衡,進(jìn)一步增加心血管疾病的風(fēng)險(xiǎn)。
[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鹵0.020)瀵圭収緇，

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