【作者】 李鑫；

【导师】 曹洁；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2019， 博士

【摘要】 背景与目的:阻塞性睡眠呼吸暂停综合征(OSAS)是以睡眠间歇性低氧为主要病理特征的睡眠呼吸疾病,此种低氧模式可以造成全身多系统损伤,其中以心血管损伤系统最为常见和严重。慢性阻塞性肺病(COPD)是以不完全可逆的气流受限为主要病理特征的进展性疾病,中重度患者可引发慢性持续低氧并导致心血管合并症的发生。呼吸重叠综合征(respiratory overlap syndrome,OS)是指OSA与COPD并存,同时存在间歇低氧和持续低氧。研究发现,其心血管损害会更为严重,死亡率也会更高。心血管疾病的发生和血管内皮的损伤明显相关,而以中性粒细胞(PMN)为代表的炎症细胞与血管内皮细胞间的交互作用成为内皮细胞损伤的关键环节。目前关于OS病理机制方面的研究较少。本研究通过间歇低氧伴持续低氧动物模型的建立,模拟OS患者间歇伴持续低氧模式。重点探讨OS模式下PMN和内皮细胞的凋亡和损伤情况,以及其基因调控机制。进而观察抗氧化或抗炎干预是否可改善PMN和内皮细胞的异常凋亡,从而减少低氧造成的损伤。本研究为揭示OS患者心血管并发症的发病机制及可行干预措施提供必要理论和实验依据,对减少OS患者心血管并发症的发生,降低OS患者的死亡率有重要的临床意义。研究内容:1.建立间歇低氧重叠熏烟动物模型,研究OS模式下大鼠PMN和内皮细胞凋亡情况及抗氧化或抗炎的干预效应。2.研究OS模式下大鼠氧化应激和炎症反应及抗氧化或抗炎的干预效应。3.研究OS模式下PMN促凋亡和抗凋亡基因的表达情况。方法:第一部分:将成年雄性Wistar大鼠48只,随机分为常氧对照组(NC组)、间歇低氧组(IH组)、熏烟暴露组(CH组),间歇低氧重叠熏烟暴露组(OS组),间歇低氧重叠熏烟暴露+Tempol干预组(OST组),间歇低氧重叠熏烟暴露+PDTC干预组(OSP组)共6组,分别给予IN、IH、CH和OS模式低氧暴露8周。分离大鼠外周血PMN和循环血内皮细胞(CECs),通过流式细胞仪检测PMN和CECs凋亡情况。应用Tunel法染色大鼠主动脉内皮,计算荧光显微镜下主动脉内皮细胞凋亡率。第二部分:利用ELISA法检测外周血中炎症指标:TNF-α、IL-6和IL-8;氧化应激指标:SOD、CAT、MDA和粘附分子ICAM-1的表达水平。第三部分:利用q RT-PCR法检测循环血中PMN促凋亡基因Bak和抗凋亡基因Mcl-1 m RNA的表达情况。结果:第一部分:1.和NC组、IH组和CH组相比,OS组大鼠外周血PMN凋亡率下降;应用抗氧化剂Tempol及炎症信号通路阻断剂PDTC干预后,和OS组相比,OST和OSP组大鼠PMN凋亡率增加(P<0.001)。2.和NC组、IH组和CH组相比,OS组大鼠循环血内皮细胞凋亡率增加;应用Tempol及PDTC干预后,和OS组相比,OST和OSP组大鼠循环血内皮细胞凋亡率减少(P<0.001)。和NC组、IH组和CH组相比,经Tunel法染色后,OS组大鼠主动脉内皮细胞凋亡率增加;应用Tempol及PDTC干预后,和OS组相比,OST和OSP组大鼠主动脉内皮细胞凋亡率减少(P<0.001)。3.双变量相关性分析显示:PMN凋亡率和内皮细胞凋亡率呈明显负相关(r=-0.854,P=0.000)。第二部分:1.和NC组、IH组和CH组相比,OS组大鼠ICAM-1、TNF-α、IL-6、IL-8和MDA表达水平升高,SOD、CAT表达降低;用Tempol及PDTC干预后,和OS组相比,OST和OSP组大鼠ICAM-1、TNF-α、IL-6、IL-8和MDA表达降低,SOD、CAT表达升高(P<0.001)。2.双变量相关性分析显示:PMN凋亡率和TNF-α、IL-6、IL-8、MDA、ICAM-1的表达水平呈明显负相关,(r值分别为0.767、0.849、0.660、0.644、0.885)(P=0.000);PMN凋亡率和SOD、CAT的表达水平呈明显正相关,(r值为0.794、0.751)(P=0.000);内皮细胞凋亡率和TNF-α、IL-6、IL-8、MDA、ICAM-1的表达水平呈明显正相关,(r值分别为0.872、0.933、0.813、0.748、0.515)(P=0.000);内皮细胞凋亡率和SOD、CAT的表达水平呈明显负相关,(r值为0.773、0.757)(P=0.000)。第三部分:1.和NC组、IH组和CH组相比,OS组大鼠Bak m RNA表达水平最低,Mcl-1 m RNA表达水平最高;应用Tempol及PDTC干预后,和OS组相比,OST和OSP组大鼠Bak m RNA表达水平升高,Mcl-1 m RNA表达水平降低(P<0.01)。2.和NC组、IH组和CH组相比,OS组大鼠Mcl-1/bak表达水平最高;应用Tempol及PDTC干预后,和OS组相比,OST和OSP组大鼠Mcl-1/bak表达降低(P<0.01)。结论:1.OS大鼠较间歇低氧和熏烟大鼠存在更为明显的中性粒细胞凋亡延迟,抗氧化剂Tempol或炎症信号通路阻断剂PDTC干预后可明显减少PMN凋亡延迟。OS大鼠存在更为明显的内皮细胞凋亡和损伤,抗氧化剂Tempol或炎症信号通路阻断剂PDTC干预后可延缓内皮细胞凋亡和损伤。2.OS大鼠存在更为明显的炎症和氧化应激反应,抗氧化剂Tempol或炎症信号通路阻断剂PDTC干预后可减轻炎症和氧化应激反应。3.OS大鼠PMN促凋亡基因表达减少,抗凋亡基因表达增加,和调控PMN凋亡延迟及内皮功能损伤相关。更多还原

【Abstract】 Objective and Background:Obstructive sleep apnea syndrome(OSAS)is a sleep-disordered disease characterized by intermittent hypoxia.This hypoxic mode can cause multi-systemic damages.And the cardiovascular system injury is the most common and severe.Chronic obstructive pulmonary disease(COPD)is a progressive disease characterized by incomplete reversible airflow limitation.Moderate and severe patients can cause chronic persistent hypoxia and lead to cardiovascular complications.Respiratory overlap syndrome(OS)refers to the coexistence of OSA and COPD,with intermittent hypoxia and persistent hypoxia.It has more serious cardiovascular damage and higher mortality.The occurrence of cardiovascular disease is closely related to the damage of vascular endothelium,and the interaction between inflammatory cells represented by neutrophils(PMN)and vascular endothelial cells becomes a key link in endothelial cell injury.At present,there are few studies on the pathological mechanism of OS.In this study,the intermittent hypoxia with persistent hypoxia animal model was established to simulate the intermittent and persistent hypoxia injury model in OS patients.It focused on the apoptosis and damage of PMN and endothelial cells in OS mode,and its gene regulation mechanisms.Furthermore,it was observed whether anti-oxidation or anti-inflammatory intervention can ameliorate abnormal apoptosis of PMN and endothelial cells,thereby reducing damages caused by hypoxia.This study provides the necessary theoretical and experimental basis for revealing the pathogenesis of cardiovascular complications and feasible interventions in OS patients.It is of great clinical significance to reduce the incidence of cardiovascular complications and mortality in OS patients.Content:1.The establishment of the OS rat model with intermittent hypoxia and cigarette exposure.The research on the apoptosis of PMN and endothelial cells in OS rats and the effects of antioxidant or anti-inflammatory intervention.2.The research on the oxidative stress and inflammatory response in OS rats and the effects of antioxidant or anti-inflammatory intervention.3.The research on the expression of PMN pro-apoptotic gene and anti-apoptotic gene in OS rats and the effects of antioxidant or anti-inflammatory intervention.Methods:Section 1: Male Wistar rats(n=48)were randomly divided into normal oxygen group(NC),intermittent hypoxia group(IH),cigarette exposed group(CH),IH overlapping cigarette exposure group(OS),OS group treated with Tempol(OST),OS group treated with PDTC(OSP).The rats were given IH,CH and OS mode hypoxic exposure for 8 weeks.PMN and CECs were isolated from peripheral blood and the apoptosis of PMN and CECs were detected by flow cytometry.The aortic endothelium were stained by Tunel method,and the apoptosis of aortic endothelial cells were observed under fluorescence microscope.Section 2 :The expression level of the following inflammatory markers in peripheral blood were detected by ELISA methods: TNF-α,IL-6,IL-8,SOD,CAT,MDA and ICAM-1.Section 3: qRT-PCR method was used to detect the expression of PMN proapoptotic gene Bak and anti-apoptotic gene Mcl-1 mRNA in circulating blood.Results:Section 1:1.Compared with NC group,IH group and CH group,the apoptosis of PMN in peripheral blood of rats in OS group reduced.After the intervention of antioxidant Tempol or inflammatory signaling blocker PDTC,the apoptosis rate of PMN in OST and OSP group increased compared with that in OS group(P<0.001).2.Compared with NC group,IH group and CH group,apoptosis of circulating blood endothelial cells in OS group increased.Compared with OS group,the apoptosis rate of circulating blood endothelial cells in OST and OSP groups decreased after the intervention of Tempol or PDTC(P<0.001).Compared with NC group,IH group and CH group,the apoptosis rate of aortic endothelial cells in OS group increased after Tunel staining.Compared with OS group,the apoptosis rate of circulating blood endothelial cells in OST and OSP groups decreased after the intervention of Tempol and PDTC(P<0.001).3.Bivariate correlation analysis showed that the apoptosis rate of PMN was significant negatively correlated with that of endothelial cells(r=-0.854,P=0.000).Section 2:1.Compared with NC group,IH group and CH group,the expression levels of ICAM-1,TNF-α,IL-6,IL-8 and MDA were increased in OS group,while the expression levels of SOD and CAT were decreased.After the intervention of Tempol or PDTC,the expression levels of ICAM-1,TNF-α,IL-6,IL-8 and MDA were reduced in OST and OSP groups,while the expression levels of SOD and CAT were increased(P<0.001).2.Bivariate correlation analysis showed that there was a significant negative correlation between the apoptosis rate of PMN and the expression levels of TNF-α,IL-6,IL-8,MDA,and ICAM-1(r values were 0.767,0.849,0.660,0.644,and 0.885,respectively)(P=0.000).The apoptosis rate of PMN was positively correlated with the expression levels of SOD and CAT(r values were 0.794,0.751)(P=0.000).There was a significant positive correlation between the apoptosis rate of endothelial cells and the expression levels of TNF-α,IL-6,IL-8,MDA,and ICAM-1(r values were 0.872,0.933,0.813,0.748,and 0.515,respectively)(P=0.000).The apoptosis rate of endothelial cells was negatively correlated with the expression levels of SOD and CAT(r values were 0.773,0.757)(P=0.000).Section 3:1.Compared with NC group,IH group and CH group,the expression level of Bak mRNA in OS group was the lowest,and the expression level of Mcl-1 mRNA was the highest.After the intervention with Tempol or PDTC,the expression level of Bak mRNA and Mcl-1 mRNA in OST and OSP groups were increased and decreased compared with that in OS group(P<0.01).2.Compared with NC group,IH group and CH group,the expression level of Mcl-1/Bak in OS group was the highest.Compared with the OS group,the expression of Mcl-1 /bak was decreased in OST and OSP groups after the intervention of Tempol or PDTC(P<0.01).Conclusion:1.Compared with the group of intermittent hypoxia and cigarette exposure,the OS rats presented more significant neutrophil apoptosis delay,which could be reduced by the intervention of antioxidant Tempol or inflammatory signaling pathway blocker PDTC.The OS rats showed more obvious apoptosis of endothelial cells and more obvious adhesion of endothelial cells.The intervention of antioxidant Tempol or inflammatory signaling blocker PDTC can delay the apoptosis and damage of endothelial cells.2.More significant inflammatory and oxidative stress responses were observed in the OS rats.Intervention with antioxidant Tempol or inflammatory signaling pathway blocker PDTC can significantly reduce inflammatory and oxidative stress responses.3.The expression of PMN pro-apoptotic genes decreased in OS rats,and the expression of anti-apoptotic genes increased,which was associated with the regulation of PMN apoptosis delay and endothelial cells damage.更多还原