睡眠呼吸暂停模式间歇低氧大鼠炎症损伤机制的研究

发布时间：2018-01-27 07:43

本文关键词： 间歇低氧阻塞性睡眠呼吸暂停综合征炎症内皮功能障碍核因子kappa B　出处：《天津医科大学》2012年博士论文　论文类型：学位论文

【摘要】：阻塞性睡眠呼吸暂停综合征(Obstructive Sleep Apnea Syndrome, OSAS)是一种发病率很高的疾病,并且是多种心血管疾病的独立危险因素。OSAS合并心血管并发症的发病机制尚不十分明确,可能是多因素疾病。炎症过程导致的内皮功能障碍可能在OSAS的心血管疾病发病机制中发挥了核心作用。其中OSAS独特的低氧模式-间歇低氧(Intermittent Hypoxia, IH),被认为是炎症过程重要的始动因素。但是间歇低氧导致炎症的机制以及低氧程度与炎症反应之间的关系尚不十分清楚。鉴于上述原因,我们建立了不同程度间歇低氧大鼠模型,观察血清循环炎性标志物水平及心脏和血管组织氧敏感转录因子活性,以探讨间歇低氧及低氧程度与炎症反应之间的关系,进一步明确IH诱发内皮功能障碍的机制,为将来临床抗炎治疗提供实验数据和理论依据。内容 1.不同程度间歇低氧大鼠模型的建立 2.不同程度间歇低氧大鼠血清循环炎性标志物的研究 3.间歇低氧与心血管系统氧敏感转录因子的研究方法第一部分：建立不同程度间歇低氧大鼠动物模型,160只雄性Wistar大鼠随机分为5组,分别给与不同低氧环境刺激,5%,7.5%,10%间歇低氧组(IH-1,2,3组),10%持续低氧对照组(CH组)和间歇常氧对照组(SC组)各32只,分别于低氧暴露第2,4,6,8周随机抽取8只大鼠麻醉后处死,取血清及主动脉血管内皮和心肌组织待用。第二部分：应用ELISA法检测血清炎性细胞因子TNF-a,IL-6,IL-8,CRP和抗炎细胞因子IL-10浓度。第三部分：应用ELISA法检测大鼠心肌组织ICAM-1浓度；应用Real-timePCR法检测大鼠主动脉血管内皮和心肌组织c-fos, VEGF mRNA表达水平；Western Blot法检测血管内皮和心肌组织转录因子NF-κBP65,HIF-1α的蛋白水平。结果第一部分： 1.不同程度间歇低氧大鼠血气分析结果显示,IH-1,2,3组大鼠最低P02分别为35.6mmHg,40.3mmHg,48.8mmHg;CH组P02维持在37.4-39.6mmHg之间；SC组P02为98-102mmHg。 2.各程度IH组大鼠收缩压明显升高,4周时高于自身前水平,6周时明显高于CH组和SC组(P0.05)。第二部分： 1.各程度IH组血清炎性细胞因子TNF-a,IL-8,IL-6和CRP水平均随时间出现明显升高,抗炎因子IL-10明显下降,F值分别为14.637,6.42,43.814,3.642和5.787,P值均0.001。 2.在低氧暴露6周时,各程度IH组血清TNF-a和IL-8水平上升达到峰值,随着低氧暴露时间进一步延长,血清水平出现下降趋势,IL-10出现与之对应的变化。而IL-6、CRP水平随时间呈现持续升高的趋势。 3.在炎症反应最严重的第6周时,各IH组TNF-α、IL-8和IL-6水平均明显高于SC组(P值均0.001)且明显高于CH组(P值均0.05)。IL-10水平明显低于SC组和CH组(P值0.01和0.05)。 4.各程度间歇低氧组间比较,在低氧暴露6周时,IH-1组TNF-α和IL-8水平明显高于IH-3组,(F值分别为1.20,34.68,P值分别为0.049,0.046)。第三部分： 1.IH-1组大鼠心肌组织匀浆ICAM-1浓度明显升高,6周时达到峰值浓度,明显高于CH组和SC组(P值分别为0.009和0.000)。低氧8周时ICAM-1水平略有下降。 2.各IH组和CH组主动脉内皮细胞核内NF-κBP65蛋白水平均较SC组明显增加(P0.01)。IH组在6周时升高达到顶峰,在随后的2周内略有下降。 3.6周时IH-1,2,3组NF-κBP65蛋白水平均明显高于CH组(P=0.000,0.002,0.012)。 4.各程度间歇低氧组间比较,IH-1组NF-κBP65蛋白水平高于IH-3组(P=0.002)。 5.各实验组心肌组织HIF-1α蛋白检测均未发现目的条带,但其下游基因VEGF mRNA的内皮和心肌表达水平在IH-1组和CH组均明显增加,高于SC组,P值均小于0.05。 6.心肌组织c-fos mRNA表达水平在IH-1组和CH组明显增加,8周时明显高于SC组(P=0.000,0.005)。结论 1.不同程度间歇低氧大鼠模型可以模拟OSAS患者不同程度的睡眠低氧。 2.间歇低氧引起大鼠收缩压明显升高。 3.间歇低氧和持续低氧均可引起系统性和心血管局部的炎症反应。 4.间歇低氧引起的炎症损伤反应比持续低氧更为严重。 5.间歇低氧引起的炎症损伤有一定的低氧程度依赖性。 6.间歇低氧引起的炎症反应出现先增强后减弱的变化趋势,说明机体代偿机制和适应性反应的存在。 7.间歇低氧和持续低氧均可引起多种转录因子的激活,主要包括NF-κB,HIF-1和AP-1。 8.随着低氧时间的延长,慢性间歇低氧可能通过多种途径诱发炎症损伤反应和适应性保护反应达到新的平衡。 9.间歇低氧主要通过激活NF-κB启动和放大炎症反应,诱发内皮功能障碍可能是OSAS相关心血管疾病发病机制的重要部分。
[Abstract]:Obstructive sleep apnea syndrome (Obstructive Sleep Apnea Syndrome, OSAS) is a very high incidence of the disease, and pathogenesis of many cardiovascular diseases.OSAS independent risk factor for cardiovascular complications is still not clear, may be multifactorial disease. The inflammatory process leads to endothelial dysfunction may play a central role OSAS in the pathogenesis of cardiovascular diseases. The OSAS unique mode of hypoxia - intermittent hypoxia (Intermittent, Hypoxia, IH) is considered to be the initiating factor of inflammatory process important. But intermittent hypoxia leads the relationship between inflammation mechanism and the degree of hypoxia and inflammation remains unclear. In view of the above reasons, we established. The rat model of different degree of intermittent hypoxia, the serum circulating levels of inflammatory markers and cardiac and vascular tissue oxygen sensitive transcription factor activity, to explore To discuss the relationship between intermittent hypoxia and hypoxia and inflammatory response, further clarify the mechanism of endothelial dysfunction induced by IH, so as to provide experimental data and theoretical basis for future clinical anti-inflammatory treatment.
content
1. establishment of rat model of intermittent hypoxia in different degrees
Study of 2. serum circulating inflammatory markers in rats with different degrees of intermittent hypoxia
3. study of oxygen sensitive transcription factors in intermittent hypoxia and cardiovascular system
Method
The first part: the establishment of animal models of different degree of intermittent hypoxia in rats, 160 male Wistar rats were randomly divided into 5 groups, were given different hypoxia stimulation, 5%, 7.5%, 10% intermittent hypoxia group (IH-1,2,3 group), 10% sustained hypoxia control group (CH group) and intermittent normoxia control group (Group SC) the 32, respectively in the 2,4,6,8 week hypoxic exposure anesthesia 8 rats were randomly selected and sacrificed, endothelium and myocardium in serum and aorta for use.
The second part: the serum levels of inflammatory cytokines TNF-a, IL-6, IL-8, CRP and anti-inflammatory cytokine IL-10 were detected by ELISA method.
The third part: the detection of ICAM-1 concentration in rat myocardium by ELISA method; Real-timePCR method was used to detect the rat aortic endothelial and myocardial tissue c-fos, VEGF expression level of mRNA; Western Blot method to detect vascular endothelial and myocardial tissue NF- transcription factor kappa BP65 alpha, HIF-1 protein level.
Result
Part one:
1. the blood gas analysis of rats with different degrees of intermittent hypoxia showed that the lowest P02 in group IH-1,2,3 was 35.6mmHg, 40.3mmHg, 48.8mmHg, P02 in CH group was between 37.4-39.6mmHg, SC group P02 was 98-102mmHg..
2. the systolic pressure of rats in group IH was significantly higher than that at 4 weeks. At 6 weeks, it was significantly higher than that in group CH and group SC (P0.05).
The second part:
1. the levels of serum inflammatory cytokines TNF-a, IL-8, IL-6 and CRP in all IH groups increased significantly with time. The IL-10 of inflammatory factors decreased significantly, F values were 14.637,6.42,43.814,3.642 and 5.787, P values were all 0.001..
2., at 6 weeks after hypoxia exposure, the serum TNF-a and IL-8 levels increased to a peak at all levels of IH group. With the further prolongation of hypoxia exposure time, serum level decreased and IL-10 changed correspondingly. However, the level of IL-6 and CRP continued to increase with time.
3., at the sixth week of the most severe inflammatory reaction, the levels of TNF-, IL-8 and IL-6 in the IH group were significantly higher than those in the SC group (P = 0.001), which was significantly higher than that in the CH group (P value 0.05). The.IL-10 level was significantly lower than that in the SC group and the CH group (0.01 and 0.05 of the 0.01).
4. compared with the intermittent hypoxia group, the levels of TNF- and IL-8 in group IH-1 were significantly higher than those in group IH-3 at 6 weeks after hypoxia exposure (F values were 1.20,34.68 and P values were 0.049,0.046).
The third part:
The concentration of 1.IH-1 in myocardial tissue homogenate ICAM-1 group rats increased significantly, 6 weeks to reach the peak concentration, was significantly higher than that of CH group and SC group (P = 0.009 and 0). 8 weeks of hypoxia ICAM-1 level decreased slightly.
2. of the IH group and CH group of aortic endothelial cell nucleus NF- kappa BP65 protein levels were significantly increased than the SC group (P0.01).IH group peaked at 6 weeks, a decline in the subsequent 2 week 4.
At 3.6 weeks, the level of NF- kappa BP65 protein in group IH-1,2,3 was significantly higher than that in group CH (P=0.000,0.002,0.012).
4. the level of NF- kappa BP65 protein in group IH-1 was higher than that in group IH-3 (P=0.002).
5. there was no target band in the detection of HIF-1 alpha protein in each experimental group, but the expression level of VEGF mRNA downstream of endothelial cells and myocardium in IH-1 group and CH group increased significantly, higher than that in SC group, P value was less than 0.05..
6. the expression level of c-fos mRNA in myocardial tissue was significantly increased in group IH-1 and CH group, and was significantly higher than that in group SC at 8 weeks (P=0.000,0.005).
conclusion
1. different degrees of intermittent hypoxia rat model can simulate different levels of sleep hypoxia in OSAS patients.
The systolic pressure of rats increased significantly in 2. intermittent hypoxia.
3. intermittent hypoxia and continuous hypoxia can cause systemic and cardiovascular local inflammatory reactions.
The inflammatory reaction caused by intermittent hypoxia in 4. is more serious than that of continuous hypoxia.
5. the inflammatory damage caused by intermittent hypoxia is dependent on a certain degree of hypoxia.
6. the changes in the inflammatory response caused by intermittent hypoxia first increased and then weakened, indicating the existence of the body's compensatory mechanism and the adaptive response.
7. the activation of multiple transcription factors can be induced by intermittent hypoxia and continuous hypoxia, including NF- kappa B, HIF-1 and AP-1.
8. with the prolongation of hypoxic time, chronic intermittent hypoxia may induce inflammatory and adaptive protection responses to a new balance through a variety of pathways.
9. intermittent hypoxia mainly activates and activates NF- kappa B and induces endothelial dysfunction, which may be an important part of the pathogenesis of OSAS related cardiovascular diseases.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R766

【参考文献】