【作者】 董丽霞；

【导师】 陈宝元；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 博士

【摘要】 研究背景与目的:睡眠呼吸暂停综合征的病理损伤核心因素是体内长期存在的间歇低氧/再氧合环境,因其显著的多系统损害被认为是全身性疾病。在OSAS诸多靶器官损害中,血管内皮功能障碍发挥着关键作用,血管内皮结构与功能的损害与动脉粥样硬化、内皮依赖性血管舒张功能障碍、凝血机制紊乱密切相关。但是慢性间歇低氧导致内皮损伤的机制,低氧程度、低氧频率与内皮损伤程度的相关性尚未完全清楚。鉴于上述原因,我们建立了间歇低氧人脐静脉内皮细胞模型,探讨不同间歇低氧模式（不同IH程度、不同IH频率、不同IH程度累加不同IH频率）下血管内皮细胞分泌ROS、氧化还原敏感的转录因子及相应炎症介质的变化,以及他们之间的相互关系,进一步明确间歇低氧导致内皮损伤的机制;同时探讨不同类型的CIH所导致的内皮细胞的氧化应激与炎症状态的差异,从而为临床治疗提供实验数据和理论依据。内容:1.不同模式CIH暴露人脐静脉内皮细胞氧化应激反应的研究2.不同模式CIH、ROS与氧化还原敏感转录因子的研究3.不同CIH与黏附分子及肿瘤坏死因子α的研究方法:应用计算机程控装置输送不同氧浓度的气体制造内皮细胞间歇低氧暴露内环境,分为三种间歇低氧程度,五种低氧频率,以间歇正常氧为对照。应用ELISA分别检测细胞分泌的培基中GSH-PX与MDA; ICAM-1与TNF-α; Western-Blot检测细胞质中HIF-1,细胞质和细胞核中的NF-κB浓度,EMSA检测NF-κB的DNA结合活性。结果:第一部分:1.在特定IH条件下,不同IH频率暴露MDA结果不同（F=652.88, P =0.00）。绘制MDA随IH频率变化的曲线,呈山峰状,非直线性。顶点在20次/H;就抗氧化能力而言,不同IH频率暴露GSH-PX结果不同（F=12300,P=0.00）,大多减低,9/h组GSH-PX明显高于对照组和其他间歇低氧暴露和持续低氧组。2.在固定IH频率12次/H时,不同IH程度,MDA（F=503.06, P=0.00）, GSH（F=23390, P=0.00）结果不同。3.三种低氧程度与五个IH频率共15种组合的暴露条件下MDA、GSH-PX浓度的检测,经过析因设计资料的方差分析后,结果显示两个变量均存在IH程度与IH频率的交互作用, MDA（F=94.14, P=0.00）,GSH（F=173.18, P=0.00）。4.双变量相关分析法显示:相关系数r=-0.52, P=0.00,提示MDA与GSH-PX呈负相关关系。第二部分:1. CIH与对照组及SH比较统计结果显示:胞质NF-κB的含量（F=1.71,P=0.19）,各组间无统计学差异;胞核NF-κB的含量（F=15206, P=0.00）,各组间具有统计学差异,CIH暴露组明显高于对照组及持续低氧组。在固定IH1.5%O₂条件下,IH频率不同的各组胞核的NF-κB的水平不同（F=74.42, P=0.00）,具有统计学差异。2. EMSA NF-κB DNA结合活性检测结果显示三种低氧程度与五个IH频率共15种组合的暴露组间存在差异（F=33.1 1, P=0.00）,析因设计资料的方差分析显示存在交互作用。3. 15种CIH模式的NF-κB DNA结合活性与相应的GSH-PX相关性分析（r=-0. 51, P=0. 00）,存在负相关。4.12次/h, 1. 5%的间歇低氧暴露模式,HIF-1不表达。5.1.5%O₂暴露不同频率c-fosmRNA的水平不同（F=67.05, P=0.00）,随频率增加出现增加后下降趋势。频率为12/h组与其他各组比较,P<0.01。第三部分:1.固定频率12次/h的间歇低氧暴露TNF-α和ICAM-1均高于对照组及持续低氧组。（F=668.41, P=0.00）2.间歇低氧程度与频率TNF-α（F=168. 06, P=0. 00）和ICAM-1（F=39. 06,P=0. 00）均表现出了明显交互作用。3.在炎症因子与NF-κB的相关性分析中发现,TNF-α（r=0. 19, P=0. 22）和ICAM-1（ r=0. 27, P=0. 06）与NF-κB DNA结合活性的相关性不明显。结论:1.本研究发现血管内皮细胞不同模式IH暴露下,表现出氧化应激与炎症损伤,但也发现部分CIH模具有保护效应。2.不同模式CIH对于氧化应激和转录因子激活、炎症反应均存在差异,显示了IH程度与频率的交互效应。3. CIH选择性激活炎症通道,未见适应性通道的激活4.氧化应激损伤可能作为始动因素进而导致炎症反应,NF-κB与氧化应激等共同影响炎症因子的释放本课题的创新点:1.目前尚未见离体人血管内皮细胞进行不同模式CIH暴露研究报告;2.目前未见不同间歇低氧程度和频率内皮损伤机制关系的研究报告;3.不同的IH/ROX模式导致内皮细胞氧化应激与炎症,不同程度、频率IH暴露下,内皮细胞表现出了损伤效应。部分CIH模式有促使抗氧化能力升高的效应.思考:1.深入研究氧化应激与炎症反应的相互作用2.继续抗氧化与抗炎干预性研究3.尝试寻求机体适应性的CIH模式更多还原

【Abstract】 Background and objective:OSAS is seemed to be a systemic disease.People with chronic IH are prone tocardiac and vascular damages.Much attention has been paid to endothelialdysfunction,which is regarded as the contributors to vascular diseases.Endothelialdysfunction can induce atherosclerosis,impairment of endothelium-dependentvasodilatation and blood coagulation disorder.The long-standing IH/ROX is the keypathology of Obstructive sleep apnea syndrome （OSAS）,Chronic intermittenthypoxemia may be responsible for triggering endothelial dysfunction in OSAS.Butthe mechanism about it is not very clear,so we estimate a model to mime theoccurrence of intermittent hypoxia,investigating the difference of oxidative andinflammatory level of endothelial cell which is exposed in various intermittenthypoxia environments,The purpose is to approach the possible mechanism of CIHrelated vascular endothelial impairment.Contents:1.The research about CIH and oxidative stress,the HUVECs exposed in differentCIH styles2.The research about CIH and oxidoreduction sensitive transcription factors3.The research about CIH and inflammatory factors.Methods:Set up a different intermittent hypoxia environment of HUVECs by computercontrol.Divided different groups by three level oxygen concentration and five IHfrequency.Investigate the level of GSH-PX,MDA,TNF-αand ICAM-1 by ELISAmethod;Detect NF-κB and HIF-1 by Western-Blot,the activity of NF-κB DNAcombination by EMSA.Results: The first part:1.In different IH frequency exposure,the levels of MDA were higher than controland SH group.For GSH-PX,the levels were different to different IH frequency,most of them were higher than control and SH group,but for an exceptionalresult,in the group of 9/h,the level of it was increased markedly than controlgroup.2.In different IH degree,the levels of MDA and GSH-PX were all increased.3.We found the interplay between IH degree and frequency by analysis the 15 CIHstyles4.In the relational analysis of GSH-PX and MDA,we found the negativecorrelation between them.The second part:1.For NF-κB,the concentration in cell plasma had no difference with control andSH group,but in nucleolus,the NF-κB concentration was higher evidently thanother groups.2.For the detecting of the DNA combinational activity of NF-κB,we also foundthe difference in 15 groups and the interplaying between IH degree andfrequency.3.The result of correlation analysis between GSH-PX and the combinationalactivity of NF-κB,the index is r=-0.51,P=0.00,significant negative correlation.4.In the expression of CIH （IH=1.5% O₂,frequency 12/h）,we did not find theexpression of HIF-1.5.In the expression of the c-fox mRNA,we found the difference expressionbetween the different IH frequency,the group frequency 12/h was higher thanothers.The third part:1.In the fix up frequency of 12/h,we found the difference levels of TNF-αandICAM-1 between CIH with control and SH groups.2.In the testing ofTNF-αand ICAM-1 in 15 groups,we found the interplay ofIH degree and frequency too. 3.In the correlation analysis of combinational activity of NF-κB to TNF-αandICAM-1,we did not detect significant correlations.Conclusions:1.To HUVECs,the exposure to different CIH styles prone to oxidative stressand inflammatory damages,but we found a exceptional type,which hadprotecting effects.2.The exposure of different CIH styles were with different degrees of vascularendothelial impairment,but the variable trend was irregular.We found theevident interplay effects between IH degree and IH frequency.3.CIH active the inflammatory transcriptional factors selectively,we did notdetect the active of adaptable pathway HIF-1.4.In our research,we consider oxidative stress may be the original factor,which can induce the activation of NF-κB,the link between CIH andinflammatory factors.NF-κB and oxidative stress co-operate to effect therelease of inflammatory factors.更多还原