【摘要】：研究目的與背景：阻塞性睡眠呼吸暫停綜合征(OSAS)是指在睡眠期反復(fù)發(fā)生上氣道阻塞并引起呼吸暫停的一種常見病。該病睡眠時(shí)反復(fù)出現(xiàn)呼吸暫停,伴隨不同程度的間歇低氧(IH)。OSAS是一種全身系統(tǒng)性疾病,炎癥和氧化應(yīng)激是其主要特征,對心血管系統(tǒng)的損害已得到國內(nèi)外呼吸和心血管領(lǐng)域的普遍認(rèn)可和關(guān)注。白細(xì)胞與內(nèi)皮細(xì)胞黏附可導(dǎo)致內(nèi)皮細(xì)胞損傷,在內(nèi)皮功能障礙導(dǎo)致的心血管合并癥中發(fā)揮重要作用。目前認(rèn)為OSAS模式IH激活的中性粒細(xì)胞及中性粒細(xì)胞與血管內(nèi)皮細(xì)胞的相互作用可引起一系列炎癥介質(zhì)的變化,但迄今IH對淋巴細(xì)胞作用研究甚少,淋巴細(xì)胞與內(nèi)皮細(xì)胞的黏附及相互作用導(dǎo)致內(nèi)皮損傷等機(jī)制尚不清楚。研究表明在OSAS中,IH能夠激活單核細(xì)胞,進(jìn)而促使黏附分子和ROS的產(chǎn)生且單核細(xì)胞對內(nèi)皮細(xì)胞的黏附活性顯著增強(qiáng),提示在OSAS損傷內(nèi)皮細(xì)胞機(jī)制中單核細(xì)胞具有重要的功能。基于以上結(jié)果我們假設(shè)IH誘導(dǎo)大鼠循環(huán)血中淋巴細(xì)胞的激活,激活的淋巴細(xì)胞與內(nèi)皮細(xì)胞黏附增加,并進(jìn)一步激活內(nèi)皮細(xì)胞,釋放促炎細(xì)胞因子TNF-α、IL-8、CRP和ICAM-1等,同時(shí)氧化及抗氧化狀態(tài)失衡,炎性介質(zhì)以接觸依賴的方式誘導(dǎo)內(nèi)皮細(xì)胞的損傷,多種轉(zhuǎn)錄因子參與內(nèi)皮細(xì)胞損傷及凋亡。為此,本研究模擬OSAS建立IH大鼠模型,觀察IH模型大鼠淋巴細(xì)胞及亞型的凋亡,檢測淋巴細(xì)胞與內(nèi)皮細(xì)胞共培養(yǎng)炎性因子及氧化應(yīng)激的水平,同時(shí)檢測參與內(nèi)皮細(xì)胞凋亡及通路的信號蛋白,最后探討抗氧化劑干預(yù)效果,為臨床OSAS合并癥的發(fā)病機(jī)制提供理論依據(jù)。內(nèi)容1.IH暴露下大鼠淋巴細(xì)胞亞群凋亡狀態(tài)及抗氧化劑Tempol干預(yù)研究2.IH暴露下大鼠淋巴細(xì)胞與血管內(nèi)皮細(xì)胞共培養(yǎng)后炎癥及氧化應(yīng)激程度,及內(nèi)皮細(xì)胞凋亡機(jī)制的研究。方法1.將56只雄性Wistar大鼠隨機(jī)分為①常氧對照組(NC);②IH 4周組(IH4);③IH 6周組(IH6);④早期抗氧化干預(yù)(IH6T)組;⑤早期生理鹽水干預(yù)(IH6N);⑥晚期抗氧化干預(yù)組(IH6T2);⑦晚期生理鹽水干預(yù)(IH6N2)。每組8只,IH暴露環(huán)境最低氧濃度均為5%,AHI 30/h。應(yīng)用Tempol從暴露開始時(shí)及暴露4周后分別進(jìn)行早期(IH6T組)和晚期(IH6T2組)干預(yù),予生理鹽水干預(yù)作對照。暴露結(jié)束后,各組大鼠均麻醉后腹主動脈取血,分離純化淋巴細(xì)胞,無血清RPMI-1640培養(yǎng)基中重懸,用抗體標(biāo)記淋巴細(xì)胞亞群,流式檢測凋亡2.使用正常大鼠主動脈內(nèi)皮細(xì)胞,細(xì)胞分為常氧對照組及IH組(IH暴露5h),淋巴細(xì)胞選用大鼠IH 6周組(IH6)、早期干預(yù)(IH6T)及常氧對照組(NC),直接接種于已含內(nèi)皮細(xì)胞的孔板中,共培養(yǎng)分為6組：①常氧大鼠淋巴細(xì)胞與常氧內(nèi)皮組(NC+NE)；②常氧大鼠淋巴細(xì)胞與IH內(nèi)皮組(NC+IHE)；③IH6周大鼠淋巴細(xì)胞與常氧內(nèi)皮組(IH6+NE)；④IH6周大鼠淋巴細(xì)胞與IH內(nèi)皮組(IH6+IHE)；⑤抗氧化干預(yù)大鼠淋巴細(xì)胞與常氧內(nèi)皮(IH6T+NE)；⑥抗氧化干預(yù)大鼠淋巴細(xì)胞與IH內(nèi)皮組(IH6T+IHE).將共培養(yǎng)板置于細(xì)胞培養(yǎng)箱內(nèi)共培養(yǎng)4h,取上清液離心后分裝待檢測,內(nèi)皮細(xì)胞提取蛋白及mRNA.3.采用ELISA法檢測各上清液CRP.TNF-α、IL-8、ICAM-1、MDA、CAT及SOD的水平,用蛋白印跡法檢測內(nèi)皮細(xì)胞內(nèi)Caspase3、NF-κB P65、Bcl-2及Bax蛋白的表達(dá)。Real-time PCR法檢測內(nèi)皮細(xì)胞內(nèi)NADPH P22、C-FOS、HIF-1α以及MAPK P38 mRNA表達(dá)水平。結(jié)果第一部分IH6組、IH4組與NC組比較,IH6組與IH4組比較,CD4、CD8淋巴細(xì)胞凋亡減少,B、NK淋巴細(xì)胞凋亡增多。IH6T、IH6T2組與IH6比較,IH6T與IH6T2比較,CD4、CD8淋巴細(xì)胞凋亡增加,B、NK淋巴細(xì)胞凋亡減少。IH6TIH6T2組與NC比較,CD4、CD8淋巴細(xì)胞凋亡減少,B、NK淋巴細(xì)胞凋亡增多,F值分別為：15.57(CD4)；24.79(CD8)；18.158(B)；21.94(NK)。第二部分結(jié)果1.淋巴細(xì)胞與內(nèi)皮共培養(yǎng)后,IH6+IHE.H6+NE組與NC+NE組,IH6+IHE分別與IH6+NE.NC+IHE相比,NC+IHE組與NC+NE組相比,共培養(yǎng)液CRP、TNF-α、IL-8、ICAM-1及MDA的水平明顯升高,但SOD和CAT明顯下降；IH6T+NE與IH6+NE組,IH6T+IHE與IH6+IHE比較,TNF-α、IL-8、ICAM-1、CRP及MDA的水平明顯下降,但SOD和CAT明顯上升；但I(xiàn)H6T+NE組與NC+NE組,IH6T+IH組E與NC+IHE組比較,TNF-α、IL-8、ICAM-1、CRP及MDA的水平仍明顯升高,但SOD和CAT明顯下降；F值分別為26.30(TNF-α)、 14.048(IL-8)、26.96(ICAM-1)、17.477(CRP)、76.75(MDA)、18.76(SOD)及 28.30(CAT).2.淋巴細(xì)胞與內(nèi)皮共培養(yǎng)后,IH6+IHE組、H6+NE組與NC+NE組,IH6+IHE組分別與IH6+NE組、NC+IHE相比,NC+IHE組與NC+NE組相比,內(nèi)皮細(xì)胞表達(dá)NF-kB P65、Caspase-3、Bax蛋白增加,BCL-2蛋白表達(dá)減少；IH6T+NE組與IH6+NE組,IH6T+IHE組與IH6+IHE比較,內(nèi)皮細(xì)胞表達(dá)NF-kB P65、 Caspase-3、Bax蛋白減少,BCL-2蛋白表達(dá)增加；但I(xiàn)H6T+NE組與NC+NE組,IH6T+IHE組與NC+IHE比較,內(nèi)皮細(xì)胞表達(dá)NF-kB P65、Caspase-3、Bax仍蛋白增加,BCL-2蛋白表達(dá)減少；F值分別為82.65(NF-kB P65).37.68(Caspase-3)、 41.009(Bax)、51.72(BCL-2)、60.681(BCL-2/Bax)。3.淋巴細(xì)胞與內(nèi)皮細(xì)胞共培養(yǎng)后,IH6+IHE組、H6+NE組與NC+NE組,IH6+IHE組分別與IH6+NE組、NC+IHE組相比,NC+IHE組與NC+NE組相比較,內(nèi)皮細(xì)胞表達(dá)NADP P22 mRNA、C-FOS mRNA、HIF-1α、MAPK P38 mRNA增多；IH6T+NE組與IH6+NE組,IH6T+IEH組與IH6+IHE組比較,內(nèi)皮細(xì)胞表達(dá)NADPH P22、C-FOS、HIF-1α和MAPK P38 mRNA減少；但I(xiàn)H6T+NE組與NC+NE組,IH6T+IHE組與NC+IHE組比較,內(nèi)皮細(xì)胞表達(dá) NADP P22 mRNA、C-FOS mRNA、HIF-1α、MAPK P38 mRNA仍增多；F值分別為27.64(NADPH P22).72.772(C-FOS)、30.04(HIF-1α)、43.84(MAPK P38)。結(jié)論1.IH暴露大鼠體內(nèi)的淋巴細(xì)胞凋亡狀態(tài)被改變,并可導(dǎo)致免疫失衡,抗氧化劑干預(yù)能夠改善IH的損傷作用。2.IH暴露大鼠淋巴細(xì)胞與血管內(nèi)皮細(xì)胞相互作用,導(dǎo)致氧化/抗氧化失衡,血管內(nèi)皮細(xì)胞釋放炎癥因子及細(xì)胞損傷與凋亡增加。Ⅲ暴露淋巴細(xì)胞誘導(dǎo)血管內(nèi)皮細(xì)胞凋亡,進(jìn)而導(dǎo)致內(nèi)皮功能障礙,在IH導(dǎo)致心血管合并癥的發(fā)病機(jī)制中發(fā)揮重要作用。3.多種凋亡相關(guān)信號蛋白參與相關(guān)血管內(nèi)皮細(xì)胞凋亡過程,促進(jìn)相關(guān)血管內(nèi)皮細(xì)胞凋亡4.抗氧化干預(yù)能夠改善IH損傷性炎性反應(yīng)和氧化／抗氧化失衡,且早期干預(yù)效果明顯。為抗氧化干預(yù)預(yù)防和治療間歇低氧引發(fā)的心腦血管合并癥提供研究理論依據(jù)。
[Abstract]:Objective and background: obstructive sleep apnea syndrome (OSAS) is a common disease caused by recurrent airway obstruction and apnea during sleep. The recurrent apnea during sleep is recurrent, with varying degrees of intermittent hypoxia (IH).OSAS, a systemic disease, and inflammation and oxidative stress. Characteristics, the damage to the cardiovascular system has been widely recognized both at home and abroad. Adhesion of leukocytes and endothelial cells can lead to endothelial cell damage and play an important role in cardiovascular complications caused by endothelial dysfunction. Currently, OSAS mode IH activated neutrophils and neutrophils and neutrophils are considered. The interaction of vascular endothelial cells can cause a series of changes in inflammatory mediators, but so far little research has been made on the action of IH on lymphocytes. The mechanisms of adhesion and interaction of lymphocytes and endothelium are not clear. The study shows that in OSAS, IH can activate monocytes and then induce adhesion molecules and ROS production. The adhesion activity of mononuclear cells to endothelial cells increased significantly, suggesting that monocytes have important functions in the mechanism of OSAS damage to endothelial cells. Based on the above results, we hypothesized that IH induced activation of lymphocytes in circulating blood in rats, activated lymphocytes and endothelial cells, and further activated endothelial cells to release the cells. Inflammatory cytokine TNF- alpha, IL-8, CRP and ICAM-1, and oxidative and antioxidant state imbalance, inflammatory mediators induce endothelial cell damage in contact dependent manner, and multiple transcription factors participate in endothelial cell injury and apoptosis. Therefore, this study simulated OSAS to establish IH rat model, observe the apoptosis of lymphocyte and subtype of IH model rats, and detect the apoptosis of lymphocyte and subtype in IH model rats. The levels of inflammatory factors and oxidative stress were co cultured with endothelial cells, and the signal proteins involved in the apoptosis and pathways involved in endothelial cells were detected. Finally, the effect of antioxidant intervention was explored to provide a theoretical basis for the pathogenesis of clinical OSAS complication. Content of apoptosis and antioxidant activity of lymphocyte subsets in rats exposed to 1.IH Tempol intervention to study the study of inflammation and oxidative stress after co culture of lymphocytes and vascular endothelial cells in 2.IH exposed rats and the mechanism of endothelial cell apoptosis. Method 1. 56 male Wistar rats were randomly divided into 1 normal oxygen control group (NC); (2) IH 4 week group (IH4); IH 6 weeks group (IH6); (4) early antioxidant intervention (IH6T) group; 5 Stage physiological saline intervention (IH6N); (6) advanced antioxidation intervention group (IH6T2); terminal physiological saline intervention (IH6N2). 8 rats in each group were 5%, and AHI 30/h. applied Tempol from the beginning of exposure and 4 weeks after exposure to the early (IH6T) and late (IH6T2 group) intervention, respectively, to the physiological saline dry advance control. Exposure to the end of the exposure to the end of the exposure. After anesthesia, all rats were taken blood from the abdominal aorta, isolated and purified the lymphocytes, the serum free RPMI-1640 medium was suspended, the lymphocyte subgroup was marked with antibodies, and the flow cytometry was used to detect the apoptosis 2. of the normal rat aortic endothelial cells. The cells were divided into the normal oxygen control group and the IH group (IH exposure 5H), the lymphocyte was selected in the IH 6 week group (IH6), and the early stage (IH6). The intervention (IH6T) and the normal oxygen control group (NC) were directly inoculated to the Kong Banzhong containing endothelial cells. The co culture was divided into 6 groups: (1) the lymphocytes of the normal oxygen rats and the normoxic endothelial group (NC+NE); (2) the lymphocytes of the normal oxygen rats and the IH endothelium group (NC+IHE); (3) the lymphatic cells and the normal oxygen endothelium (IH6+NE) in the IH6 week rats; (4) the lymphocyte and IH in IH6 week rats and IH Endothelial group (IH6+IHE); (5) antioxidative intervention in rat lymphocytes and oxygen endothelium (IH6T+NE); (6) the lymphocyte and IH endothelial group (IH6T+IHE) in antioxidant intervention rats (IH6T+IHE). Co culture plate was placed in cell culture box to co culture 4h, and the supernatant was centrifuged after centrifugation to be detected. The endothelial cells extracted protein and mRNA.3. were used to detect the supernatant by ELISA method. The levels of CRP.TNF- alpha, IL-8, ICAM-1, MDA, CAT and SOD were detected by Western blot, and the expressions of NF- kappa B P65, Bcl-2 and Bax proteins were detected in endothelial cells. Lymphocyte apoptosis decreased, B, NK lymphocyte apoptosis increased.IH6T, IH6T2 group compared with IH6, IH6T and IH6T2, CD4, CD8 lymphocyte apoptosis increased, B, NK lymphocyte apoptosis decreased compared to.IH6TIH6T2 group and apoptosis, lymphoid cell apoptosis increased, respectively: 15.57; 24.79; 18.158; 21.94 (NK). Second part of result 1. lymphocyte and endothelium co culture, IH6+IHE.H6+NE group and NC+NE group, IH6+IHE compared with IH6+NE.NC+IHE, NC+IHE group compared with NC+NE group, TNF- alpha, IL-8, ICAM-1 and MDA. 8, ICAM-1, CRP and MDA significantly decreased, but SOD and CAT increased obviously, but IH6T+NE and NC+NE groups, IH6T+IH group E and NC+IHE groups, TNF- alpha, IL-8, SOD, and decreased significantly decreased, respectively, 14.048, 26.96, 17.477, 76.75, 18.76, and 28.30. (CAT) after co culture of.2. lymphocyte and endothelium, group IH6+IHE, H6+NE group and NC+NE group, IH6+IHE group were compared with IH6+NE group, NC+IHE, NC+IHE group and NC+NE group, the expression of NF-kB P65 was compared with NC+NE group. 5, Caspase-3, Bax protein decreased, and the expression of BCL-2 protein increased; but in group IH6T+NE and NC+NE, IH6T+IHE group was compared with NC+IHE, and endothelial cells expressed NF-kB P65, Caspase-3, Bax protein and decreased expression of BCL-2 protein; 41.009, 51.72, 60.681, respectively. After co culture, IH6+IHE group, H6+NE group and NC+NE group, IH6+IHE group compared with group IH6+NE, NC+IHE group, NC+IHE group compared with NC+NE group, endothelial cells expressed NADP P22 mRNA. And MAPK P38 mRNA decreased, but in group IH6T+NE and NC+NE group, IH6T+IHE group and NC+IHE group, the endothelial cells expressed NADP P22 mRNA, C-FOS mRNA, which were 27.64, 30.04 (alpha), 43.84 (respectively). Conclusion the apoptosis state of lymphocytes in exposed rats was changed, Immune imbalance can lead to immune imbalance, antioxidant intervention can improve the IH damage effect,.2.IH exposure of lymphocytes and vascular endothelial cells interaction, resulting in oxidative / antioxidant imbalance, vascular endothelial cells release inflammatory factors and cell damage and apoptosis increase. Skin dysfunction plays an important role in the pathogenesis of IH associated with cardiovascular complications..3. multiple apoptosis related signaling proteins participate in the apoptosis process of vascular endothelial cells, and the 4. antioxidant intervention to promote vascular endothelial cell apoptosis can improve the IH damage inflammatory response and oxygenation / antioxidant imbalance, and the early intervention effect is clear. It provides a theoretical basis for the prevention and treatment of cardiovascular and cerebrovascular complications induced by intermittent hypoxia.
【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】：2014
【分類號】：R766

【共引文獻(xiàn)】

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2 Aylin Karalezli;Fatma Corak Eroglu;Tulay Kivanc;Rusina Dogan;;Evaluation of choroidal thickness using spectral-domain optical coherence tomography in patients with severe obstructive sleep apnea syndrome: a comparative study[J];International Journal of Ophthalmology;2014年06期

3 王釗;姚曉光;汪迎春;李南方;;冠狀動脈狹窄<50%的夜間胸痛與阻塞性睡眠呼吸暫停低通氣綜合征的關(guān)系[J];中國動脈硬化雜志;2014年02期

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5 辛亞男;郭陽;;OSAHS合并高血壓患者的臨床特征分析[J];臨床肺科雜志;2014年12期

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8 寇育樂;解寶泉;王紅陽;張嘉賓;譚曦舒;張敏;;慢性間歇低氧對大鼠肺組織損傷及依達(dá)拉奉的干預(yù)作用[J];臨床耳鼻咽喉頭頸外科雜志;2015年19期

9 許雙朝;姜領(lǐng);;高壓氧對糖尿病腎病患者血管內(nèi)皮功能的影響[J];實(shí)用醫(yī)學(xué)雜志;2013年21期

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