本文關(guān)鍵詞： 間歇低氧 阻塞性睡眠呼吸暫停綜合征 炎癥 內(nèi)皮功能障礙核因子kappa B　出處：《天津醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：阻塞性睡眠呼吸暫停綜合征(Obstructive Sleep Apnea Syndrome, OSAS)是一種發(fā)病率很高的疾病,并且是多種心血管疾病的獨(dú)立危險因素。OSAS合并心血管并發(fā)癥的發(fā)病機(jī)制尚不十分明確,可能是多因素疾病。炎癥過程導(dǎo)致的內(nèi)皮功能障礙可能在OSAS的心血管疾病發(fā)病機(jī)制中發(fā)揮了核心作用。其中OSAS獨(dú)特的低氧模式-間歇低氧(Intermittent Hypoxia, IH),被認(rèn)為是炎癥過程重要的始動因素。但是間歇低氧導(dǎo)致炎癥的機(jī)制以及低氧程度與炎癥反應(yīng)之間的關(guān)系尚不十分清楚。鑒于上述原因,我們建立了不同程度間歇低氧大鼠模型,觀察血清循環(huán)炎性標(biāo)志物水平及心臟和血管組織氧敏感轉(zhuǎn)錄因子活性,以探討間歇低氧及低氧程度與炎癥反應(yīng)之間的關(guān)系,進(jìn)一步明確IH誘發(fā)內(nèi)皮功能障礙的機(jī)制,為將來臨床抗炎治療提供實(shí)驗(yàn)數(shù)據(jù)和理論依據(jù)。 內(nèi)容 1.不同程度間歇低氧大鼠模型的建立 2.不同程度間歇低氧大鼠血清循環(huán)炎性標(biāo)志物的研究 3.間歇低氧與心血管系統(tǒng)氧敏感轉(zhuǎn)錄因子的研究 方法 第一部分：建立不同程度間歇低氧大鼠動物模型,160只雄性Wistar大鼠隨機(jī)分為5組,分別給與不同低氧環(huán)境刺激,5%,7.5%,10%間歇低氧組(IH-1,2,3組),10%持續(xù)低氧對照組(CH組)和間歇常氧對照組(SC組)各32只,分別于低氧暴露第2,4,6,8周隨機(jī)抽取8只大鼠麻醉后處死,取血清及主動脈血管內(nèi)皮和心肌組織待用。 第二部分：應(yīng)用ELISA法檢測血清炎性細(xì)胞因子TNF-a,IL-6,IL-8,CRP和抗炎細(xì)胞因子IL-10濃度。 第三部分：應(yīng)用ELISA法檢測大鼠心肌組織ICAM-1濃度；應(yīng)用Real-timePCR法檢測大鼠主動脈血管內(nèi)皮和心肌組織c-fos, VEGF mRNA表達(dá)水平；Western Blot法檢測血管內(nèi)皮和心肌組織轉(zhuǎn)錄因子NF-κBP65,HIF-1α的蛋白水平。 結(jié)果 第一部分： 1.不同程度間歇低氧大鼠血?dú)夥治鼋Y(jié)果顯示,IH-1,2,3組大鼠最低P02分別為35.6mmHg,40.3mmHg,48.8mmHg;CH組P02維持在37.4-39.6mmHg之間；SC組P02為98-102mmHg。 2.各程度IH組大鼠收縮壓明顯升高,4周時高于自身前水平,6周時明顯高于CH組和SC組(P0.05)。 第二部分： 1.各程度IH組血清炎性細(xì)胞因子TNF-a,IL-8,IL-6和CRP水平均隨時間出現(xiàn)明顯升高,抗炎因子IL-10明顯下降,F值分別為14.637,6.42,43.814,3.642和5.787,P值均0.001。 2.在低氧暴露6周時,各程度IH組血清TNF-a和IL-8水平上升達(dá)到峰值,隨著低氧暴露時間進(jìn)一步延長,血清水平出現(xiàn)下降趨勢,IL-10出現(xiàn)與之對應(yīng)的變化。而IL-6、CRP水平隨時間呈現(xiàn)持續(xù)升高的趨勢。 3.在炎癥反應(yīng)最嚴(yán)重的第6周時,各IH組TNF-α、IL-8和IL-6水平均明顯高于SC組(P值均0.001)且明顯高于CH組(P值均0.05)。IL-10水平明顯低于SC組和CH組(P值0.01和0.05)。 4.各程度間歇低氧組間比較,在低氧暴露6周時,IH-1組TNF-α和IL-8水平明顯高于IH-3組,(F值分別為1.20,34.68,P值分別為0.049,0.046)。 第三部分： 1.IH-1組大鼠心肌組織勻漿ICAM-1濃度明顯升高,6周時達(dá)到峰值濃度,明顯高于CH組和SC組(P值分別為0.009和0.000)。低氧8周時ICAM-1水平略有下降。 2.各IH組和CH組主動脈內(nèi)皮細(xì)胞核內(nèi)NF-κBP65蛋白水平均較SC組明顯增加(P0.01)。IH組在6周時升高達(dá)到頂峰,在隨后的2周內(nèi)略有下降。 3.6周時IH-1,2,3組NF-κBP65蛋白水平均明顯高于CH組(P=0.000,0.002,0.012)。 4.各程度間歇低氧組間比較,IH-1組NF-κBP65蛋白水平高于IH-3組(P=0.002)。 5.各實(shí)驗(yàn)組心肌組織HIF-1α蛋白檢測均未發(fā)現(xiàn)目的條帶,但其下游基因VEGF mRNA的內(nèi)皮和心肌表達(dá)水平在IH-1組和CH組均明顯增加,高于SC組,P值均小于0.05。 6.心肌組織c-fos mRNA表達(dá)水平在IH-1組和CH組明顯增加,8周時明顯高于SC組(P=0.000,0.005)。 結(jié)論 1.不同程度間歇低氧大鼠模型可以模擬OSAS患者不同程度的睡眠低氧。 2.間歇低氧引起大鼠收縮壓明顯升高。 3.間歇低氧和持續(xù)低氧均可引起系統(tǒng)性和心血管局部的炎癥反應(yīng)。 4.間歇低氧引起的炎癥損傷反應(yīng)比持續(xù)低氧更為嚴(yán)重。 5.間歇低氧引起的炎癥損傷有一定的低氧程度依賴性。 6.間歇低氧引起的炎癥反應(yīng)出現(xiàn)先增強(qiáng)后減弱的變化趨勢,說明機(jī)體代償機(jī)制和適應(yīng)性反應(yīng)的存在。 7.間歇低氧和持續(xù)低氧均可引起多種轉(zhuǎn)錄因子的激活,主要包括NF-κB,HIF-1和AP-1。 8.隨著低氧時間的延長,慢性間歇低氧可能通過多種途徑誘發(fā)炎癥損傷反應(yīng)和適應(yīng)性保護(hù)反應(yīng)達(dá)到新的平衡。 9.間歇低氧主要通過激活NF-κB啟動和放大炎癥反應(yīng),誘發(fā)內(nèi)皮功能障礙可能是OSAS相關(guān)心血管疾病發(fā)病機(jī)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R766

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 康進(jìn)朝,劉維永,蔡振杰,李彤;SD鼠未成熟心肌細(xì)胞缺氧/復(fù)氧期c-fos,c-myc mRNA的表達(dá)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2000年05期

2 曹卓;王良興;;CPAP治療阻塞型睡眠呼吸暫停綜合征前后患者血清IL-6和TNF-α的動態(tài)變化[J];放射免疫學(xué)雜志;2009年01期

3 張赤;c-fos原癌基因與心肌缺血再灌注[J];國外醫(yī)學(xué).麻醉學(xué)與復(fù)蘇分冊;1998年02期

4 李艷玲;陳曉輝;劉世明;鄺錦輝;曹梅;陳曉軍;張永健;蘇杭;;OSAHS患者外周血中白介素6水平及其mRNA表達(dá)的變化和意義[J];廣州醫(yī)學(xué)院學(xué)報(bào);2007年02期

5 張希龍,童茂榮,夏錫榮,施毅,曹鄂洪,張偉;鼾癥老年人睡眠呼吸暫停癥與心血管疾病的相關(guān)性[J];中華老年心腦血管病雜志;2001年03期

6 陳麗,高興玉,張俊,劉利,羅勇,呂湛,劉小華;老年高血壓肱動脈內(nèi)皮功能、頸動脈粥樣硬化與血清腫瘤壞死因子α、白介素6關(guān)系的研究[J];四川醫(yī)學(xué);2005年05期

7 樊昕;杜鳳和;田俊萍;;炎性介質(zhì)在OSAHS與冠心病關(guān)系中的作用[J];山東醫(yī)藥;2008年22期

8 鮑永霞;呂福禎;馬迎軍;;低氧對小鼠低氧誘導(dǎo)因子-1α和血管內(nèi)皮生長因子表達(dá)的影響[J];中國實(shí)用內(nèi)科雜志;2006年14期

9 楊曦明,謝印芝,聶鴻靖,尹昭云;低氧大鼠肺動脈內(nèi)皮細(xì)胞VEGF變化與PKC活性關(guān)系的探討[J];中國應(yīng)用生理學(xué)雜志;2000年03期

10 白莉,余祖濱,錢頻,錢桂生,關(guān)崧,李淑萍;SOCS 3基因?qū)θ毖醴蝿用}平滑肌細(xì)胞增殖及原癌基因mRNA表達(dá)的影響[J];中華內(nèi)科雜志;2005年01期

，

本文編號：1467982