【作者】 王强；

【导师】 刘卓拉；

【作者基本信息】 山西医科大学 ， 呼吸内科， 2010， 博士

【摘要】 研究背景：阻塞性睡眠呼吸暂停低通气综合征(obstructive sleep apnea-hyponea syndrome, OSA)是睡眠呼吸暂停综合症中最为常见的类型,具体表现为睡眠过程中咽部气道塌陷,是一种以上气道狭窄甚至完全闭塞导致的频繁呼吸暂停为特征的睡眠呼吸障碍性疾病。国外的调查显示,成年人阻塞性睡眠呼吸暂停综合征的平均发病率为4%[1],近年报道发病率有增长的趋势,患者多在40岁以上,且发病率为男性高于女性。2003年到2004年,上海[2]、承德[3]、太原[4]等城市进行了阻塞性睡眠呼吸暂停综合征的流行病学调查,累计调查170821人,平均患病率为3.5-4.8%。OSA是一种累计多系统并造成多器官损害的睡眠呼吸疾病,是高血压、冠心病、心率失常、脑卒中等多种疾病的独立危险因素,其对机体的损害还包括精神系统、泌尿生殖系统、内分泌系统、呼吸系统、消化系统、肌肉系统等全身多个系统,理论上讲,人体内凡是与氧代谢相关的部分都会被波及影响,对人类的健康和生命构成了严重的威胁。临床统计显示,未经治疗的OSA患者5年病死率达11%-13%,.呼吸紊乱指数(Apnea Hypopnea Index, AHI)高于20者8年病死率高达37%[5],全球每天约有3000人的死亡与OSA有关。OSA的主要病理损伤因素包括：睡眠结构破坏、胸腔压力异常改变和慢性缺氧,其中关键的病理损伤因素就是体内长期慢性间歇性低氧环境的存在及其通过对体内氧化应激炎症反应通路等信号网络通道的影响,造成对多个靶向器官的损伤。内源性大麻素系统是一种新近阐明的信号系统,参与了神经系统以及周围器官的多种生理功能,广泛涉及机体多种病理过程：近来研究提示内源性大麻素系统可调控下列过程：1.血管收缩；2.炎症过程；3.免疫调节；4.生殖行为及生育过程；5.血栓形成；6.通过对脑、小肠及迷走神经张力的影响直接和间接影响食欲；7.脂肪沉积,脂质在肝脏和脂肪细胞中的氧化；8.胰岛素分泌,肌肉和脂肪组织对葡萄糖的摄取；9.骨质代谢[6]。内源性大麻素系统对食欲、能量平衡、免疫调节、炎症反应、及血管张力的影响引起了广泛关注,目前认为内源性大麻素系统为发挥负性调节的内源性脂性信号系统,在维持机体稳态的调节中发挥重要作用[7]。我们推测OSA病情可能会干扰内源性大麻素系统,影响其维持机体稳态的作用。紊乱的内源性大麻素系统与其他同样受到OSA影响的氧化应激炎症反应通路等信号通道网络共同构成了OSA并发症发生发展的病理学基础。在下面的研究中我们将探讨内源性大麻素系统与OSA及其并发症的关系,验证我们的推论,为进一步阐明OSA并发症的病理生理机制,提供新思路。目的：(1)通过对正常及糖尿病人群外周血单个核细胞内源性大麻素系统组份表达量的检测,证实：①外周血单个核细胞拥有完整的内源性大麻素系统活性功能成分,为全身内源性大麻素系统的一部分；②糖尿病患者体内的内源性大麻素系统发生了变化；③外周血单个核细胞上内源性大麻素系统的活性可反应整体的病理生理变化。(2)通过分析OSA患者内源性大麻素系统组份表达的变化及其与OSA临床表现的相关性,证实：①OSA患者内源性大麻素系统发生了变化,且与OSA病情指标相关,提示OSA患者出现了内源性大麻素系统的失衡和紊乱；②内源性大麻素系统与OSA并发症存在相关性,可能与其他发生紊乱的信号系统一起构成了OSA并发症的发生发展的病理生理基础；(3)研究OSA患者睡前及晨起内源性大麻素系统组份表达的变化,进一步证实OSA夜间的病理生理变化可影响内源性大麻素系统组分的表达。方法：采用病例对照研究方法,(1)晨起抽血提取30例无心脑血管并发症临床表现的新发2型糖尿病患者及30例正常对照者的单个核细胞,运用实时定量PCR检测内源性大麻素的受体及合成降解酶类的表达水平变化,比较分析两组之间的差异；(2)选择无心脑血管并发症、糖脂代谢紊乱、感染等疾病的83例OSA患者和30例正常对照志愿者,通过问卷量化受试者临床表现,提取晨起外周血单个核细胞,运用细胞免疫组化、流式细胞术和实时定量PCR检测内源性大麻素的受体及合成降解酶类的表达水平变化,分析内源性大麻素系统组份表达的变化与OSA病情指标及临床症状的相关性。(3)睡前和晨起抽血提取24例OSA患者及18例健康志愿者单个核细胞,检测内源性大麻素系统各组分的变化,比较OSA夜间病理生理变化对内源性大麻素系统的影响。结果：(1)外周血单个核细胞检测出了内源性大麻素系统的两种受体大麻素受体1(cannabinoid receptor 1, CB1)、大麻素受体2 (cannabinoid receptor 2, CB2),两种主要配体N-花生四烯酸氨基乙醇(anandamide, AEA)和2-花生四烯酸甘油(2-arachidonoyl glycbrol,2-AG)合成和降解的关键酶：脂肪酰胺水解酶(fatty acid amide hydrolase, FAAH)、丝氨酸水解酶(monoacylglycerol lipase, MGL)、二酰基甘油脂肪酶(diacylglycerol lipase, DAGL)和N-酰基磷脂酰乙醇胺-磷酸酯水解酶(N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, NAP)清晰的扩增曲线,表明其可能拥有完整的内源性大麻素系统活性功能成分；与正常组相比糖尿病组受体CB1mRNA表达量明显上升,CB2mRNA表达量明显下降, t值和P值分别为1.54、2.76；0.05、0.01。(2)免疫组化和流式结果均提示,与正常组相比,OSA组受体CB2蛋白表达量明显升高,t值分别为0.95和1.82,其P值分别为0.008和0.049；OSA组受体CB2、内源性大麻素AEA合成酶NAPmRNA表达量明显升高,内源性大麻素2-AG主要降解酶MGLmRNA表达量明显降低,t值分别为1.97、1.99和2.06,其P值分别为0.05、0.05和0.04。(3)相关分析发现CB2、NAP两指标均与最长呼吸暂停时间及平均氧饱和度相关,相关系数和P值分别为：0.26、0.26,0.27、0.40；0.03、0.03,0.02、0.00。MGL与最长呼吸暂停时间、最低氧饱和度和平均氧饱和度均具有相关性相关系数和P值分别为-0.31、0.34、-0.38；0.01、0.00、0.00。多重线性回归分析提示CB2和NAP多重回归模型成立,微觉醒指数进入CB2回归模型,标准化回归系数为0.26,t值为2.69,P值为0.01；微觉醒指数和SaO2进入NAP模型,标准化回归系数分别为0.27和0.24,t值和P值分别为2.86,2.58；0.01,0.01。(4)出现认知功能受损OSA患者的内源性大麻素AEA主要合成酶NAPmRNA表达量明显升高,t值为2.48, P值为0.02；最低氧饱和度及微觉醒指数两组间比较有显著差异t值和P值为分别为1.79、1.69；0.05、0.05。(5)性功能受损组OSA患者内源性大麻素2-AG主要降解酶MGLmRNA表达量明显降低,t值为1.68, P值为0.05；微觉醒指数两组间比较有显著差异t值和P值为分别为1.46;0.05。(6)伴发高血压OSA患者内源性大麻素AEA主要降解酶FAAHmRNA表达量明显降低,t值为0.77, P值为0.01。微觉醒指数两组间比较有显著差异t值和P值为分别为1.68;0.04。(7)睡眠前后两个时间点两组间各指标mRNA△CT的变化趋势及比较发现,患OSA及夜间睡眠对MGL均有影响,处理主效应(OSA)(F=3.996,P=0.035)、时间主效应(夜间睡眠)(F=4.131,P=0.025)均有统计学意义,处理效应与时间效应的交互作用无统计学意义(F=0.956,P=0.338)。结论和意义：(1)人类外周血单个核细胞表达了从结合到代谢AEA和2-AG的全部生物活性物质,结果提示单个核细胞是内源性大麻素系统的组成部分。其组分表达水平变化与糖代谢紊乱相关,说明单个核细胞的内源性大麻素系统可能反应整体的生理病理状况,同时也支持内源性大麻素信号系统与代谢及能量平衡关系密切。本结果为第二部分研究提供了理论和实验基础。(2)在OSA人群中发现内源性大麻素系统的组分变化与反应OSA病情的PSG监测指标,特别是与微觉醒指数和夜间平均血氧饱和度关系密切,OSA患者内源性大麻素系统总体表现为活性增强。活性增强的内源性大麻素系统通过其广泛的负性调节作用,促使机体内环境能够重新恢复平衡。活化的内源性大麻素系统可能通过抑制外周交感神经系统的活性,降低血压,为机体再次应对应激做准备,但同时也可能促进了对认知功能和性功能的抑制。(3)晨起和睡前内源性大麻素系统各组分发生了变化,进一步证实OSA的夜间睡眠紊乱病理状态与文献报道的其他急性应激一样,均可引起以2-AG作用增强为表现的内源性大麻素系统的变化。此变化可能是机体应对应激的保守反应,并参与终止应激反应、恢复机体平衡状态。这一结果进一步支持了第二部分的结论。更多还原

【Abstract】 Background:Obstructive sleep apnea hypopnea syndrome (OSA) is a highly prevalent sleep disorder and its evidence is up to 3-4%in the population. The current knowledges on the OSA suggests that it is an independent risk factor for many kinds of diseases, such as hypertension, coronary heart disease, stroke, metabolize dysfunction, sexual dysfunction and so on. It is said that OSA would affect every physiological mechanism that correlation with oxygen metabolism. Main pathological damages of OSA include sleep structure damage, chest abnormal pressure change and chronic hypoxia, where the key factors in pathological damage is the existence of the long-term chronicintermittenthypoxia(CIH) environment and the inflammatory response pathway inside oxidatie stress signal channel network, causing damage to multiple target organs. The endocannabinoid (EC) system is an endogenous signaling system which is involved in numerous physiological functions, both in the central and peripheral nervous systems and in peripheral organs. It is composed of the cannabinoid receptors, endocannabinoids and the molecular machinery required for their synthesis and degradation. The EC system has been implicated in a wide range of pathological conditions ranging from mood and anxiety disorders, movement disorders, hypertension to the metabolize dysfunction. Now, EC system is belived to be a neuroactive lipid signaling system that functions to gate homeostasis maintainence. OSA may interfere with endocannabinoids system, and affect its steady-state function. Disorder of endocannabinoids system and other signal network influenced by OSA will constitute the pathological basis for the development of OSA complications. In the following study, we will examine the relationship between endocannabinoids system and OSA and its complications.Objective:(1) The expression of endocannabinoids system component in peripheral mononuclear cells of normal and diabetic population were tested, to confirm①peripheral mononuclear cells have complete functional components of endogenous cannabinoid system;②endogenous cannabinoid system has changed in diabetes;③the activity of endocannabinoids system on peripheral mononuclear cells can reflect the overall pathological physiological changes. (2) detected the expression of endogenous cannabinoids and its relationship with clinical manifestations in OSA patients, to confirm④endocannabinoids system changes in OSA patients;②endocannabinoids system is associated with OSA complications; (3) Through exploring the expression changes of endocannabinoid system in patients before and after sleep to further evidence the influence of OSA pathophysiology on endocannabinoid system.Methods:Cast-control studies were adopted to do the following research.①Real-time quantitative PCR was used to detect the difference of endocannabinoid system in type 2 diabetes and normal controls groups;②The differences of endocannabinoid system in OSA and normal controls groups were detected and the correlation between endocannabinoids system component changes and the clinical manifestations in OSA patients were also analysised.③Mononuclear cells were extracted from peripheral blood samples of 24 cases of OSA patients and 18 healthy volunteers before and after sleep, and changing of endocannabinoid system were also detected to explore the influence of the pathological physiology change of OSA on it.Results:1. Clear amplification curves of the two kinds of endocannabinoid receptors CB1, CB2, and FAAH, MGL, DAGL and Nape, the key-synthesis and degradation enzymes of the two main ligands endocannabinoid AEA and 2-AG, were detected from peripheral blood mononuclear cells. Indicating that mononuclear cells might have the total functional compositions of endocannabinoid system. Compared with the normal control, in diabetic group, mRNA of CB1 increased noticeable(t=1.54, P=0.05), and CB2 decreased significantly(t=2.76, P=0.01)2. Results from immunohistochemistry and flow cytometry showed that, compared with the normal group, CB2 receptor protein expression was significantly higher in OSA (t=0.95,1.82; P=0.008,0.049). In OSA group, mRNA of CB2 increased significantly(t=1.97, P=0.05); NAP, key synthase of AEA, increased significantly(t=1.99, P=0.05); MGL, main 2-AG-degrading enzyme, decreased significantly(t=2.06, P=0.04).3. Correlation analysis showed that CB2 and NAP were correlation with the longest apnea time(Tmax) and the average oxygen saturation(SaO2min). MGL was correlation with Tmax, SaO2min and the average oxygen saturation (SaO2). Multiple linear regression analysis showed that multiple regression models of CB2 and NAP were established, micro-arousal index into the CB2 regression model, standardized regression coefficient 0.26, t=2.69, P=0.01; micro-arousal index and the SaO2 into the NAP model, standardized regression coefficient respectively,0.27 and 0.24, t=2.86,2.58 and P=0.01,0.01.4. In the group of OSA patients with cognitive impairment, mRNA of NAP was significantly higher, t=2.48,P=0.02; SaO2min and arousal index between the two groups have more significant difference, t=1.79,1.69; P=0.05,0.05.5. In the group of OSA patients with impaired sexual function, mRNA of MGL was significantly reduced, t=1.68, P=0.05; micro-arousal index between the two groups were significantly different, t=1.46, P=0.05.6. In OSA patients with hypertension, mRNA of FAAH, main AEA-degrading enzyme was significantly lower, t=0.77, P=0.01. Micro-arousal index between the two groups were significantly different, t=1.68; P=0.04.7. MGL was influenced by OSA and nocturnal sleep, the treatment main effect (OSA) (F=3.996,P=0.035) and time main effect (nocturnal sleep) (F=4.131, P=0.025) were statistically significant, the interaction between treatment effect and time effect has no statistical significance (F=0.956, P=0.338).Conclusion:1. Human peripheral blood mononuclear cells expresses the full biochemical substances to bind and metabolize AEA and 2-AG The finding that human mononuclear cells show a functional "endocannabinoid system" indicates that peripheral actions of the endocannabinoids may have a role in mononuclear cells. Expression level of endocannabinoids system components changes with glucose metabolism disorder, indicating mononuclear cells may respond to physiological and pathological conditions as a whole. This section provided a theoretical and experimental basis for the following research.2. In OSA group, changes of the endocannabinoids system component correlated with PSG, especially with nocturnal arousal index and SaO2. The endocannabinoids system of OSA patients enhance overall performance of activity. Through its regulatory role in a wide range of negative, endocannabinoids system would prompt the body to regain balance. It would lower blood pressure, inhibit the activity of peripheral sympathetic nervous system, in order to cope with stress, but may also promote inhibition of cognitive and sexual function.3. Like other acute stress in the literature, nocturnal pathological state of OSA may lead to the enhanced role of 2-AG and participate in the termination of the stress response and restore the body balance. This section further supports the results of the second part.更多还原