【作者】 王林；

【导师】 朱惠莉；

【作者基本信息】 复旦大学 ， 内科学， 2012， 硕士

【摘要】 研究目的通过建立不同程度的间歇缺氧动物模型,模拟不同程度睡眠呼吸暂停低通气综合征(SAHS)患者的缺氧状态,研究不同程度间歇缺氧对大鼠脑细胞凋亡发生发展的意义；对缺氧动物模型实施21%氧干预后,观察其对脑组织病理变化的影响,加深对SAHS的认识,为早期干预SAHS病情的发生与发展提供科学依据；观察对间歇缺氧进行21%氧干预后,大鼠脑内HIF-1, iNOS表达的变化,探讨二者在间歇缺氧神经细胞凋亡中的作用,同时阐明这一变化对评估缺血性脑血管疾病的治疗价值的意义。材料与方法健康SD大鼠42只,适应性饲养1周。实验起始阶段：42只中随机选取6只作为0周的参考值(A组)。剩余36只大鼠随机分为三组,对照组(n=12)；轻度间歇缺氧组(n=12,相当于轻度OSAS, AHI=15);重度间歇缺氧组(n=12,相当于重度OSAS, AHI=40),各组以普通饲料、普通饮水饲养60天。每日9：00至16：00时将各组大鼠置于各实验箱中,共7h,16:00后使大鼠生活于大气环境中。各组在试验30天后随机取6只大鼠(BN组,BQ组,BZ组),予以取脑、原位细胞凋亡检测(TUNEL)、电镜检查,应用免疫组化,Western Blot对脑内HIF-1α, iNOS进行蛋白水平的检测。各组剩余大鼠(CN组,CQ组,CZ组)予以正常氧环境下继续饲养,30天后行上述相同检查。结果应用SPSS17.0软件进行统计分析。结果1.神经细胞形态学变化：1.1HE染色光学显微镜下,神经细胞核呈蓝黑色,胞浆呈淡红色。A组,BN组SD大鼠海马区可见数层锥体神经元,细胞形态规则；BQ, BZ各组中,从形态学观察有不同程度的改变,海马区锥体神经元出现核固缩、胞质浓缩、细胞体急剧变小等现象,以此判断可能有细胞凋亡的发生。经过21%氧干预30天后,试验结果发现：干预组(CQ, CZ组)神经细胞凋亡情况有所好转,但部分神经元细胞仍有排列不规则,形态改变的情况,未能恢复至初始水平。1.2透视电镜检查发现：经过30天间歇缺氧,间歇缺氧组的脑组织病理较正常氧组有显著变化,且重度间歇缺氧组较轻度间歇缺氧组改变更为明显,表现为大鼠脑组织神经细胞核双层膜结构破坏,部分线粒体水肿扩张,细胞质中可见空泡。经过21%氧干预30天后,干预组神经细胞凋亡情况有所好转,但部分神经细胞仍有细胞形态的变化,未能恢复至初始水平。1.3TUNEL实验显示：对照组(A组,BN组)SD大鼠海马区可见神经细胞形态规则,排列整齐紧密；缺氧组(BQ, BZ)中,被染成棕黄色或棕褐色凋亡细胞增多,以重度间歇缺氧组增多最为明显(14.83±2.48VS18.67±2.66VS32.67±4.18VS49.17±4.36)。间歇缺氧组(BQ, BZ组)明显高于对照组(A,BN组),差异有统计学意义(P<0.01)；轻度间歇缺氧组和重度间歇缺氧组之间两两比较差异有统计学意义(P<0.01)。经过21%氧干预30天后,试验结果发现：间歇缺氧干预组(CQ, CZ组)神经细胞凋亡情况有所好转(24.00±3.90VS31.67±2.50),但未能恢复至初始水平。间歇缺氧干预组(CQ, CZ组)高于对照组(A, BN, CN组),差异有统计学意义(P<0.01)；和缺氧组(BQ, BZ组)之间比较差异有统计学意义(P<0.01)。凋亡神经细胞主要集中在海马区,皮层。2. iNOS表达的比较常氧对照组(A,BN组)大鼠脑组织内散在分布少量iNOS阳性细胞(23.46±3.5501,24.17±1.4452)。在实验组,不论是轻度间歇缺氧组还是重度间歇缺氧组均可见较多的着色细胞(37.01±4.0473,53.34±8.0801)。间歇缺氧组(BQ, BZ组)明显高于对照组(A, BN组),差异有统计学意义(P<0.01)；轻度间歇缺氧组和重度间歇缺氧组之间两两比较差异有统计学意义(P<0.01)；经21%氧干预30天后,干预组(CQ, CZ组)脑内iNOS阳性染色细胞表达恢复至初始水平(24.6±8.1662,24.77±6.6268),干预组(CQ,CZ组)和间歇缺氧组(BQ,BZ组)比较差异有统计学意义,和对照组(A, BN, CN组)比较差异无统计学意义(P>0.05)。iNOS阳性染色细胞主要集中在大脑皮层及海马区,不仅见于神经元,神经胶质细胞中亦有表达。WestemBlot检测结果显示：缺氧后,实验组(BQ, BZ组)iNOS较对照组(A,BN组)表达增多,以慢性重度间歇缺氧组最为明显(140.01±1.7147VS151.52±19.9948VS376.52±38.9234VS585.17±35.1067)。间歇缺氧组(BQ, BZ组)明显高于对照组(A,BN组),差异有统计学意义(P<0.01)；轻度间歇缺氧组(BQ组)和重度间歇缺氧组(BZ组)之间比较差异有统计学意义(P<0.01)。经21%氧干预30天后,干预组(CQ, CZ组：170.59±42.5984,172.45±30.2081)和间歇缺氧组(BQ,BZ组)比较差异有统计学意义,和对照组(A, BN, CN组)比较差异无统计学意义(P>0.05),各组脑内iNOS蛋白表达恢复至初始水平。3.HIF-1α表达的比较常对照组(A, BN组)大鼠脑组织内HIF-1α呈弱表达,在间歇缺氧组(BQ,BZ组)尤其是重度间歇缺氧组可见着色细胞明显增多(12.35±4.7915VS13.55±5.7289VS27.61±4.0600VS36.81±2.4887)。间歇缺氧组(BQ, BZ组)明显高于对照组(A,BN组),差异有统计学意义(P<0.01)；轻度间歇缺氧组和重度间歇缺氧组之间比较差异有统计学意义((P<0.01)；经21%氧干预30天后,各组脑内HIF-1α阳性染色细胞表达恢复至初始水平,干预组(CQ, CZ组：13.70±5.2587,14.22±6.5862)和间歇缺氧组(BQ, BZ组)比较差异有统计学意义,和对照组(A,BN,CN组)比较差异无统计学意义(P>0.05)。HIF-1α阳性染色细胞主要集中在海马及皮层区。WesternBlot检测结果显示：缺氧后,实验组(BQ,BZ组)HIF-1α较对照组(A,BN组)表达增多,以慢性重度间歇缺氧组最为明显(112.60±11.2176VS136.26±19.994VS338.25±81.6125VS488.63±86.4614)。间歇缺氧组(BQ,BZ组)明显高于对照组(A,BN组),差异有统计学意义(P<0.01)；轻度间歇缺氧组(BQ组)和重度间歇缺氧组(BZ组)之间两两比较差异有统计学意义(P<0.01)。经21%氧干预30天后,干预组(CQ, CZ组：158.44±10.631,167.79±34.505)和间歇缺氧组(BQ, BZ组)比较差异有统计学意义,和对照组(A, BN, CN组)比较差异无统计学意义(P>0.05),各组脑内HIF-1α蛋白表达恢复至初始水平。结论1.间歇缺氧易诱导脑内特定部位神经细胞凋亡,主要表现为皮层和海马区神经元细胞凋亡增加,这可能是SAHS导致认知功能障碍的主要原因。2.不同缺氧方式所导致神经细胞凋亡的程度是不同的,说明神经细胞的凋亡不仅仅是由缺氧本身所致,而且与缺氧的方式有关。3.间歇缺氧有效刺激海马区iNOS和HIF-1α的蛋白水平高表达,二者共同参与了神经系统缺氧应答反应。不同程度缺氧方式所导致神经细胞凋亡的程度是不同的,其HIF-1α及iNOS在脑内的蛋白表达也不同。4.缺氧干预后,脑组织损伤有一定程度的改善,可能与机体内氧自由基代谢恢复平衡有关。缺氧程度不同,氧化应激强度不同,早期纠正缺氧,改善氧化应激状态,可促进神经细胞损伤修复。更多还原

【Abstract】 ObjectiveThrough the establishment of the different degrees of intermittent hypoxia animal models to simulate the hypoxia status of different degrees of sleep apnea hypopnea syndrome (S AHS) patients, we learned about the significance of the different degrees of intermittent hypoxia on cerebral apoptosis. After21%oxygen intervention on an animal model of intermittent hypoxia, we observed the impact of pathological changes in rat brain to deepen the understanding of SAHS, in order to provide the scientific basis for early intervention in patients with SAHS development. We observed the changes of HIF-la and iNOS expression in the rat brain after21%oxygen intervention, in order to explore their role in neuronal apoptosis and clarify the significance on assessing the value of treatment of ischemic cerebral vascular diseases.Methods42healthy SD rats were adaptively fed for one week. Experimental start-up phase:6rats were randomly selected from42as the reference value of0weeks (A group). The remaining36rats were randomly divided into three groups:control group (n=12), mild intermittent hypoxia group (n=12, equivalent to mild OSAS AHI=15), severe intermittent hypoxia group (n=12, equivalent to severe OSAS AHI=40). The rats fed for60days with ordinary feed, drinking. Rats in each group are placed in the experimental box daily from9:00to16:00, a total of7h. After16:00, they lived in the atmosphere. Each group was randomly selected6rats (BN, BQ, BZ group) to be taken the brain, in situ apoptosis detection (TUNEL), electron microscopy, the brain HIF-la, iNOS by immunohistochemistry, Western blot for the detection of protein levels. The remaining rats in each group (CN, CQ, CZ group) were reared under normal oxygen environment. And after30days they underwent the same examination. Statistical analysis was performed by SPSS17.0software.Results1. Morphological changes of nerve cells:1.1The nucleus was blue-black, and the cytoplasm pale was red by HE staining under a light microscope. In the control group (A, BN group), the morphology of the nerve cell in the hippocampus of rats was very regular and neat. There are different degrees of change in the hypoxia group (BQ, BZ group) from the morphological observation, which can determine the occurrence of apoptosis. After21%oxygen intervention for30days, the results showed that:neuronal apoptosis situation in the intervention group (CQ, CZ group) had improved. However, parts of the nerve cells were still irregular, morphological changes, and failed to recover to the initial level.1.2Perspective electron microscope examination revealed:After30days of intermittent hypoxia, there were significant changes in brain tissue pathology between intermittent hypoxia group and normal oxygen group, and severe intermittent hypoxia group changed more noticeably. We observed that the nerve nucleus double membrane structure of brain tissue damaged, part of the mitochondrial appeared edema expansion, and there were vacuoles in the cytoplasm. After21%oxygen intervention for30days, neuronal apoptosis situation in the intervention group (CQ, CZ group) had improved. However, parts of the nerve cells still changed in cell morphology, and failed to recover to the initial level.1.3TUNEL assay showed:In control group, the morphology of nerve cell in the hippocampus of rats was very regular and neat. In hypoxia group (BQ, BZ group), the number of apoptosis cells that were stained brown or tan was high. Severe intermittent hypoxia group increased most obviously (14.83±2.48VS18.67±2.66VS32.67±4.18VS49.17±4.36).The number of neuronal apoptosis in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the test results showed that:the situation of neuronal apoptosis in intermittent hypoxia intervention group (CQ, CZ group) had improved,(24.00±3.90VS31.67±2.50), but did not return to the initial level. The number of neuronal apoptosis in intermittent hypoxia intervention group (CQ, CZ group) was higher than that in the control group (A, BN, CN group), the difference was statistically significant (P<0.01). The difference between the intermittent hypoxia intervention group (CQ, CZ group) and the hypoxia group (BQ, BZ group) was statistically significant (P<0.01). Apoptosis of nerve cells was mainly concentrated in the hippocampus and cortex.2. Comparison of the expression of iNOS:Normal rat brain tissue of oxygen in the control group scattered in the small number of iNOS positive cells (23.46±3.5501,24,17±1.4452). In the experimental group, whether mild intermittent hypoxia group or severe intermittent hypoxia group showed more coloring cells (37.01±4.0473,53.34±8.0801). The number of iNOS positive cells in intermittent hypoxia group (BQ, BZ group) was significantly higher than those in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, iNOS positive staining cells expression in the intervention group (CQ, CZ group) returned to the initial level (24.6±8.1662,24.77±6.6268). The difference between the intervention group (CQ, CZ group) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with the intervention group (P>0.05). The iNOS positive staining cells were mainly concentrated in the cerebral cortex and hippocampus. They were not only found in neurons but also glial cells.WesternBlot test results showed:After hypoxia, the iNOS expression in experimental group (BQ, BZ group) increased compared with the control group (A, BN group), and severe intermittent hypoxia group increased most noticeably(140.01±1.7147VS151.52±19.9948VS376.52±38.9234VS585.17±35.1067). The iNOS expression in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the difference of iNOS expression between intervention group (CQ, CZ group:170.59±42.5984,172.45±30.2081)) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with intervention group (P>0.05). The expression of iNOS protein in each group returned to the initial level.3. Comparison of the expression of HIF-1α:Normal rat brain tissue of oxygen in the control group (A, BN group) showed weak HIF-la expression. The stained cells in the intermittent hypoxia group (BQ, BZ group), especially in severe intermittent hypoxia group were significantly increased (12.35±4.7915VS13.55±5.7289VS27.61±4.0600VS36.81±2.4887). The HIF-la expression in intermittent hypoxia group (BQ, BZ group) was higher than that in the control group (A, BN group), and the difference was statistically significant (P <0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the HIF-la expression in the intervention group (CQ, CZ group) returned to the initial level (13.70±5.2587,14.22±6.5862). The difference between the intervention group (CQ, CZ group) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with the intervention group (P>0.05). The HIF-la positive staining cells were mainly concentrated in the cerebral cortex and hippocampus.WesternBlot test results showed:After hypoxia, the HIF-1α expression in experimental group (BQ, BZ group) increased compared with the control group (A, BN group), and severe intermittent hypoxia group increased most obviously C112.60d±11.2176VS136.26±19.994VS338.25±81.6125VS488.63α86.4614). The HIF-1α expression in intermittent hypoxia group (BQ, BZ group) was significantly higher than that in the control group (A, BN group), and the difference was statistically significant (P<0.01). The difference between mild intermittent hypoxia and severe intermittent hypoxia group was statistically significant (P<0.01). After21%oxygen intervention for30days, the difference of HIF-la expression between intervention group (CQ, CZ group:158.44±10.631,167.79±34.505) and intermittent hypoxia group (BQ, BZ group) was statistically significant, and the control group (A, BN, CN group) showed no significant difference with intervention group (P>0.05). The expression of HIF-la protein in each group returned to the initial level.Conclusions1. Intermittent hypoxia could undoubtedly induce nerve cells apoptosis in the specific area of the brain, mainly for the increase in neuronal apoptosis in the cortex and hippocampus, which might be the main reason for OSAHS lead to cognitive dysfunction.2. Different degrees of hypoxia could cause different levels of apoptosis of neurons, which illustrates the neuronal apoptosis was not only caused by hypoxia itself, but also the way of hypoxia.3. Intermittent hypoxia could effectively stimulate the hippocampus protein with high levels of expression of iNOS and HIF-la. Both of them participated in the nervous system hypoxia response. Different levels of hypoxia could cause atypical apoptosis of neuronal cells, and HIF-la and iNOS protein expression in the brain were also different.4. After hypoxia intervention, brain tissue injury had a certain degree of improvement, which was possibly related to the balance of metabolism of oxygen free radicals in the body. Oxidative stress intensity varied with the hypoxia extent. Correcting hypoxia and improving oxidative stress early could promote nerve cell injury and repair.更多还原