本文選題：阻塞性睡眠呼吸暫停綜合征 + 間歇低氧��； 參考：《天津醫(yī)科大學》2012年博士論文

【摘要】：阻塞性睡眠呼吸暫停綜合征(OSAS)是一種伴隨心血管疾病嚴重后果的常見疾病。OSAS心血管并發(fā)癥的發(fā)病機制尚不明確,可能是涉及各種不同機制的多因素過程。OSAS的病理生理特點為間歇低氧,這種低氧再氧合類似于缺血再灌注,會產(chǎn)生炎癥反應和組織細胞損傷。因此導致血管內(nèi)皮損傷和功能紊亂的炎癥過程在OSAS心血管并發(fā)癥的發(fā)病機制中可能發(fā)揮了關(guān)鍵作用。另外,各種心血管疾病的最終結(jié)局為心室重構(gòu)和心力衰竭,心肌細胞凋亡是心室重構(gòu)和心力衰竭發(fā)生的重要細胞分子學基礎,而細胞凋亡的過程實際上是caspases不可逆有限水解底物的級聯(lián)放大反應過程,caspase-3是caspases級聯(lián)“瀑布”下游最關(guān)鍵的凋亡蛋白酶。因此,本研究建立不同頻率間歇低氧動物模型,觀察血清循環(huán)炎性標志物水平及心肌組織不同轉(zhuǎn)錄因子活性,探討間歇低氧及低氧頻率與炎癥反應之間的關(guān)系,明確IH誘發(fā)炎癥損傷的機制；觀察IH條件下心肌細胞caspase-3蛋白表達水平,探討IH誘發(fā)心臟損傷的細胞學機制；并應用抗氧化劑干預抑制炎癥反應,為OSAS心血管并發(fā)癥的預防和治療提供新的方向和實驗依據(jù)。 內(nèi)容 1.不同頻率間歇低氧大鼠炎癥損傷機制的研究 2.抗氧化劑Tempol對間歇低氧大鼠炎癥損傷的干預效應 3.間歇低氧大鼠心肌細胞caspase-3蛋白表達水平及抗氧化劑Tempol的干預效應 方法 第一部分：將成年雄性Wistar大鼠分別暴露于不同頻率IH(IH1組：10次/h、IH2組：20次/h、IH3組：30次/h、IH4組：40次/h)和持續(xù)常氧(SC組)環(huán)境下,并采用標準飼養(yǎng)(NC)組進行對照,共暴露6周。應用ELISA法檢測血清炎性標志物TNF-α、IL-6、IL-8、CRP、ICAM-1、VEGF和抗炎細胞因子IL-10濃度；應用Western Blot法檢測心肌細胞核內(nèi)轉(zhuǎn)錄因子NF-κBP65、心肌組織HIF-1α的蛋白水平,Real-time PCR法檢測大鼠心肌組織c-fos mRNA表達水平。 第二部分：將IH(頻率為30次/h)暴露的成年雄性Wistar大鼠分成兩組,應用Tempol分別從暴露開始時及暴露4周后進行早期(]H3T0組)和晚期(IH3T組)干預,共暴露6周。觀察上述炎癥指標及不同轉(zhuǎn)錄因子水平的變化情況,并與IH3組和SC組進行比較。 第三部分：對IH3T0組大鼠,應用免疫組化法測定心肌細胞caspase-3蛋白水平,并與IH3組和SC組進行比較。 結(jié)果 第一部分： 1、與SC組、NC組比較,各頻率IH組血清炎性標志物TNF-αIL.8、IL-6、CRP、ICAM-1和VEGF水平明顯升高,抗炎因子IL.10明顯降低,F值分別為9.679、24.461、19.359、30.278、27.318、22.564和20.594,P值均為0.000。 2、與SC組、NC組比較,各頻率IH組心肌細胞核內(nèi)NF-κBP65、心肌組織HIF.1α、c-fos mRNA水平明顯升高,F值分別為35.089、24.934和22.950,P值均為0.000。 3、不同頻率IH組間比較,隨頻率增高,TNF-α、IL-8、IL-6、CRP和ICAM-1水平以及NF-κBP65、c-fos mRNA水平逐漸升高(11-13組與IH1組比較均P0.01),IL.10逐漸降低(IH3組與IH1組比較P0.01)。但IH3組與IH4組間上述指標沒有差異(P0.05),峰值(或谷值)在頻率為30次/h時出現(xiàn)。NF-κBP65水平與TNF-α、IL-8、IL-6、CRP和ICAM-1水平呈正相關(guān)(r值分別為0.519、0.574、0.608、0.659和0.576,P值分別為0.002、0.001、0.000、0.000和0.003)；與IL-10水平呈負相關(guān)(r=-0.618,P=0.000)。 4、不同頻率IH組間比較,隨頻率增高,VEGF水平及HIF.1α水平持續(xù)升高。HIF.1α水平與VEGF水平呈正相關(guān)(r=0.661,P=0.000)。 第二部分： 1、IH3T0組和IH3T組上述炎癥標志物及轉(zhuǎn)錄因子水平低于IH3組,抗炎因子水平高于IH3組。 2、IH3T0組和IH3T組上述炎癥標志物、抗炎因子及轉(zhuǎn)錄因子水平與SC組比較差異有統(tǒng)計學意義(P0.05)。 第三部分： 1、與SC組相比,IH3組心肌細胞caspase-3蛋白水平顯著增高(P0.01)。 2、IH3T0組心肌細胞caspase-3蛋白水平均明顯低于IH3組(P0.01)。 3、IH3組心肌細胞caspase-3表達水平與NF-κd3P65蛋白水平呈正相關(guān)(r=0.864,P值分別為0.006)。 結(jié)論 1.IH可以導致炎癥和抗炎系統(tǒng)失衡,從而引起系統(tǒng)性和心臟組織局部炎癥損傷。 2.IH引起的炎癥損傷有一定的間歇低氧頻率依賴性。但炎癥反應與間歇低氧頻率之間并不呈線性關(guān)系,炎癥反應隨頻率增高逐漸增強,到一定頻率后不再增高,說明機體代償機制和適應性反應的存在。 3.IH可引起不同轉(zhuǎn)錄因子的激活,主要包括NF-κB, HIF-1α和AP-1,其中中低度IH頻率優(yōu)勢激活NF-κB、AP-1介導的炎癥通路,啟動并放大炎癥反應；而過高IH頻率則優(yōu)勢激活HIF-1α介導的適應性通路,這有可能是機體的保護性機制。 4.1H可能通過活化NF-κB,轉(zhuǎn)錄調(diào)節(jié)促凋亡基因的表達,介導細胞凋亡通路中間蛋白caspase-3的活化。 5.抗氧化劑通過調(diào)節(jié)氧化/抗氧化平衡來調(diào)節(jié)炎癥/抗炎系統(tǒng)平衡,可以減輕IH誘導的炎癥損傷,抑制心肌細胞凋亡,對IH損傷有保護效應。
[Abstract]:Obstructive sleep apnea syndrome (OSAS) is a common disease associated with serious cardiovascular disease, the pathogenesis of.OSAS cardiovascular complications is not clear. It may be the pathophysiological characteristics of.OSAS, which involves various mechanisms of various mechanisms, which is characterized by intermittent hypoxia. This hypoxic reoxygenation is similar to that of ischemia reperfusion. Inflammatory reactions and tissue cell damage. Therefore, the inflammatory processes that lead to vascular endothelial damage and dysfunction may play a key role in the pathogenesis of OSAS cardiovascular complications. In addition, the final outcome of various cardiovascular diseases is ventricular remodeling and heart failure, and myocardial apoptosis is the reconstitution of ventricular remodeling and heart failure. The process of cell apoptosis is in fact the cascade amplification reaction process of caspases irreversible limited hydrolytic substrates, and caspase-3 is the most critical apoptotic protease in the cascade cascade of cascades in the caspases cascade. Therefore, this study establishes a different frequency intermittent hypoxia animal model to observe the level of serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers and to observe the level of the serum circulating inflammatory markers. The activity of different transcription factors in myocardium was used to explore the relationship between intermittent hypoxia and hypoxia frequency and inflammatory response, and to clarify the mechanism of IH induced inflammatory damage. The expression of caspase-3 protein in cardiac myocytes under IH conditions was observed and the cytological mechanism of IH induced heart damage should be explored, and the anti inflammatory intervention should be used to inhibit the inflammatory reaction, which was the OSAS heart. It provides a new direction and experimental basis for prevention and treatment of vascular complications.
content
1. the mechanism of inflammatory injury induced by intermittent hypoxia in rats at different frequencies.
2. intervention effect of antioxidant Tempol on inflammatory injury induced by intermittent hypoxia in rats
3. the expression level of caspase-3 protein in myocardial cells of intermittent hypoxia rats and the intervention effect of antioxidant Tempol
Method
Part 1: adult male Wistar rats were exposed to different frequencies of IH (group IH1: 10 times /h, IH2 group: 20 /h, IH3 group, 30 /h, IH4 group: 40 /h) and constant oxygen (SC group) environment, and the standard feeding (NC) group was exposed for 6 weeks. The concentration of F and anti inflammatory cytokine IL-10, the Western Blot method was used to detect the transcription factor NF- kappa BP65, the protein level of HIF-1 a in myocardium, and Real-time PCR method was used to detect c-fos mRNA expression level in rat myocardium.
The second part: the adult male Wistar rats exposed to IH (frequency 30 times /h) were divided into two groups. Tempol was exposed to early (]H3T0) and late (IH3T) for 6 weeks from exposure to exposure and 4 weeks after exposure. The changes of the above inflammatory indices and different transcriptional levels were observed and compared with those in the IH3 group and the SC group.
The third part: in group IH3T0, the level of caspase-3 protein in myocardial cells was detected by immunohistochemistry, and compared with group IH3 and SC.
Result
Part one:
1, compared with group SC and group NC, the levels of serum inflammatory markers, TNF- a IL.8, IL-6, CRP, ICAM-1 and VEGF were significantly increased in each frequency IH group, and the anti inflammatory factor IL.10 decreased obviously, and F was 9.679,24.461,19.359,30.278,27.318,22.564 and 20.594.
2, compared with group SC and group NC, NF- kappa BP65, HIF.1 A and c-fos mRNA in myocardial nuclei of group IH were significantly increased, F values were 35.089,24.934 and 22.950 respectively, P values were 0.000..
3, compared with the frequency of IH, the level of TNF- alpha, IL-8, IL-6, CRP and ICAM-1, NF- kappa BP65, c-fos mRNA increased gradually with the frequency of different frequencies, and the level of c-fos mRNA increased gradually (compared with the IH1 group). There is a positive correlation between the level of.NF- kappa BP65 and the levels of TNF- alpha, IL-8, IL-6, CRP and ICAM-1 (R values are 0.519,0.574,0.608,0.659 and 0.576 respectively, P values are 0.002,0.001,0.000,0.000 and 0.003 respectively), and are negatively correlated with IL-10 levels.
4, compared with the IH group, with the increase of frequency, the level of VEGF and HIF.1 alpha continued to rise. The level of.HIF.1 alpha was positively correlated with the level of VEGF (r=0.661, P=0.000).
The second part:
1, the levels of inflammatory markers and transcription factors in group IH3T0 and group IH3T were lower than those in group IH3, and the levels of anti-inflammatory factors were higher than those in group IH3.
2, the levels of inflammatory markers, anti-inflammatory factors and transcription factors in group IH3T0 and group IH3T were significantly different from those in group SC (P0.05).
The third part:
1, compared with group SC, the level of caspase-3 protein in myocardial cells of IH3 group was significantly higher (P0.01).
2, the level of caspase-3 protein in myocardial cells of group IH3T0 was significantly lower than that of group IH3 (P0.01).
3, the expression level of Caspase-3 in myocardial cells of IH3 group was positively correlated with the level of NF- kappa d3P65 protein (r=0.864, P value was 0.006).
conclusion
1.IH can lead to imbalance between inflammatory and anti-inflammatory systems, resulting in systemic and cardiac tissue inflammatory injury.
The inflammatory injury caused by 2.IH has a certain frequency dependence of intermittent hypoxia. However, there is no linear relationship between the inflammatory response and the frequency of intermittent hypoxia. The inflammatory response increases gradually with the increase of frequency, and no longer increases after a certain frequency, indicating the survival of the body's compensatory mechanism and the adaptive response.
3.IH can cause activation of different transcription factors, mainly including NF- kappa B, HIF-1 alpha and AP-1, in which the frequency of middle and low IH activates NF- kappa B, AP-1 mediated inflammatory pathways, initiates and amplifies the inflammatory response, while the frequency of high IH activates the adaptive pathway mediated by HIF-1 alpha, which may be the protective mechanism of the organism.
4.1H may regulate the expression of Pro apoptotic genes through activation of NF- kappa B, and mediate the activation of intermediate protein caspase-3 in apoptotic pathway.
5. antioxidants regulate inflammatory / anti-inflammatory balance by regulating oxidation / antioxidant balance, which can reduce the inflammatory damage induced by IH, inhibit the apoptosis of myocardial cells and protect the IH damage.

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

【引證文獻】

相關(guān)碩士學位論文 前1條

1 戎元元;術(shù)側(cè)肺輔助小潮氣量IPPV對周圍型肺癌開胸患者氧合及炎性因子的影響[D];河北醫(yī)科大學;2014年

，

本文編號：1872814