OSAS模式IH對小鼠肺纖維化的影響及其機制的初步研究
發(fā)布時間：2018-08-09 17:06
【摘要】：阻塞性睡眠呼吸暫停綜合征(obstructive sleep apnea syndrome,OSAS)是一種以睡眠過程中反復(fù)發(fā)生上氣道部分或者完全阻塞為特點導(dǎo)致睡眠結(jié)構(gòu)紊亂的慢性睡眠呼吸系統(tǒng)疾病,其在成年人的患病率為2%~4%,未經(jīng)治療的OSAS患者5年病死率達11%~13%,嚴重威脅著患者的健康和生命。間歇低氧(intermittent hypoxia,IH)是OSAS獨特的病理生理學(xué)改變,其特點為低氧-復(fù)氧間斷發(fā)生,類似于缺血/再灌注損傷,被認為與OSAS多種并發(fā)癥如:心腦血管疾病、代謝紊亂疾病、認知功能障礙、生殖泌尿功能異常和癌癥等有關(guān),對機體的危害是不容小覷的。近幾年,OSAS與肺間質(zhì)纖維化的聯(lián)系逐漸被研究人員發(fā)現(xiàn),肺間質(zhì)纖維化是一種進行性發(fā)展、致死性的、病因不明的間質(zhì)性肺疾病,它是由纖維母細胞/肌纖母細胞(fibroblasts/myofibroblasts)大量聚集和膠原(collagen)等胞外基質(zhì)(extracellular matrix,ECM)過度沉積造成的。肺纖維化確診后平均存活期為2年,5年生存率為30%~50%,病死率高,預(yù)后差,目前臨床缺乏有效治療方法。OSAS是一種系統(tǒng)性的疾病,它可以合并多種疾病,而氧化應(yīng)激是OSAS與肺間質(zhì)纖維化的共同作用機制。本研究將要初步探討OSAS模式IH對肺纖維化的影響,并討論氧化應(yīng)激對在其中所產(chǎn)生的的影響,為進一步臨床研究提供實驗基礎(chǔ)和理論依據(jù)。研究目的1、探討OSAS模式IH對博來霉素誘導(dǎo)的小鼠肺纖維化的影響。2、探討OSAS模式IH對小鼠肺成纖維細胞活化和胞外基質(zhì)分泌的影響。內(nèi)容1.OSAS模式IH對博來霉素誘導(dǎo)的小鼠肺纖維化的影響。2.OSAS模式IH對小鼠肺成纖維細胞活化和胞外基質(zhì)分泌的影響方法建立博來霉素誘導(dǎo)的小鼠肺纖維化的模型,后隨機分成3組,N組(常氧組),IH組(間歇低氧組)以及IH+T組(Tempol干預(yù)組)。對于IH+T組,每天于IH處理前半小時予Tempol腹腔注射,將3組小鼠分別放置在自制IH暴露容器內(nèi),在10:00-16:00的時段內(nèi)分別向N組持續(xù)輸送壓縮空氣,向IH組和IH+T組間斷輸送氧氣和氮氣的混合氣體,Western blot法檢測α-SMA,另于細胞培養(yǎng)箱內(nèi)培養(yǎng)成纖維細胞可傳代細胞株MLg細胞,待細胞長至80-90%融合時,接種于培養(yǎng)皿內(nèi),培養(yǎng)48h后,將調(diào)整好的肺成纖維細胞MLg隨機分成5組,將4組置于IH實驗艙內(nèi),隨機選擇1組在IH處理8小時前,進行Tempol的干預(yù),通過計算機程控裝置分別給予OSAS模式IH刺激,1h、4h、8h,及T+8h,將對照組細胞放置在培養(yǎng)箱內(nèi)培養(yǎng)24h,然后分別收集間歇低氧組和對照組細胞及上清,通過q RT-PCR法檢測α-SMA,Col1a1的表達;Western blot法檢測α-SMA,type I collagen(COL1)蛋白的表達。另統(tǒng)計分析采用SPSS16.0軟件包,數(shù)據(jù)以x±s表示.多組間比較采用單因素方差分析(One-Way ANOVA),P0.05,差異顯著,P0.01,差異極顯著。結(jié)果1.OSAS模式IH對博來霉素誘導(dǎo)的小鼠肺纖維化模型中肺組織α-SMA蛋白表達水平的影響及Tempol干預(yù)對α-SMA蛋白表達水平的影響Western blot分析結(jié)果表明:在博來霉素誘導(dǎo)的小鼠肺纖維化模型中肺組織α-SMA蛋白IH組的表達水平較常氧對照組明顯增高,差異具備統(tǒng)計學(xué)意義(P0.05),IH+T組α-SMA蛋白的表達水平較IH組降低,但高于常氧對照組,差異具備統(tǒng)計學(xué)意義(P0.05)。2.不同間歇低氧時間下肺MLg成纖維細胞中α-SMA表達水平的影響以及Tempol干預(yù)對α-SMA蛋白表達水平的影響:q RT-PCR結(jié)果表明:IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞中α-SMA m RNA的相對表達水平較常氧對照組明顯增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg細胞中α-SMA m RNA的相對表達水平隨IH時間延長而表達增多(F=104.3 P0.05),并且在IH8h達到高峰(P0.01)。Western blot分析結(jié)果表明:IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞中α-SMA蛋白的表達水平較常氧對照組增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞α-SMA蛋白的表達水平隨IH時間延長而表達增多(P0.05),并且在IH8h達到高峰(P0.05)。IH8h+Tα-SMA蛋白的表達水平較IH1h,IH4h,IH8h降低,也較N組降低(P0.01)。3.不同間歇低氧時間下肺MLg成纖維細胞中COL1表達水平的影響以及Tempol干預(yù)對COL1蛋白表達水平的影響:q RT-PCR結(jié)果表明:IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞中COL1 m RNA相對表達水平較常氧對照組明顯增高(P0.05),IH1h,IH4h,IH8h細胞中COL1m RNA的相對表達水平隨IH時間延長而表達增多(F=220.3 P0.05),并且在IH8h組達到高峰(P0.01)。Western blot分析結(jié)果表明:IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞COL1蛋白的表達水平較常氧對照組增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg成纖維細胞COL1蛋白的表達水平隨IH時間延長而表達增多(P0.05),并且在IH8h達到高峰P0.05)。IH8h+T COL1蛋白蛋白的表達水平較IH1h,IH4h,IH8h降低,但稍高于N組(P0.01)。結(jié)論1.在OSA模式IH可以加重博來霉素誘導(dǎo)的小鼠肺纖維化;抗氧化劑Tempol的干預(yù)可以減弱OSAS模式下IH對博來霉素誘導(dǎo)的小鼠肺纖維化的促進作用。2.OSAS模式IH可以促進肺成纖維細胞活化和胞外基質(zhì)分泌3.抗氧化劑Tempol的干預(yù)可以減弱OSAS模式下IH對肺成纖維細胞活化和跑外基質(zhì)分泌的促進作用;4.氧化應(yīng)激反應(yīng)在OSA模式IH促進肺纖維化的過程中有重要的調(diào)節(jié)作用。
[Abstract]:Obstructive sleep apnea syndrome (OSAS) is a chronic sleep respiratory disease, which is characterized by repeated upper airway or complete obstruction of the upper airway during sleep, with a prevalence rate of 2%~ 4% in adults and a 5 year mortality of 11%~13% in untreated OSAS patients. Intermittent hypoxia (IH) is a unique pathophysiological change of OSAS, which is characterized by hypoxic reoxygenation, similar to ischemia / reperfusion injury, and is considered to be associated with a variety of OSAS complications such as cardiovascular and cerebrovascular diseases, metabolic disorders, cognitive dysfunction, and genitourinary dysfunction. In recent years, the relationship between OSAS and pulmonary fibrosis has been gradually discovered by researchers. Pulmonary fibrosis is a progressive, fatal, interstitial lung disease with unknown etiology, which is a large accumulation of fibroblast / myofibroblast (fibroblasts/myofibroblasts). The average survival time of pulmonary fibrosis was 2 years after diagnosis, the average survival rate of pulmonary fibrosis was 2 years, the 5 year survival rate was 30%~50%, the mortality rate was high, and the prognosis was poor. At present, the clinical lack of effective treatment,.OSAS is a systemic disease, it can merge a variety of diseases, and oxidative stress is OSAS. The common mechanism of pulmonary fibrosis. This study will preliminarily discuss the effect of OSAS model IH on pulmonary fibrosis and discuss the effect of oxidative stress on it, and provide experimental basis and theoretical basis for further clinical study. Objective 1 to explore the effect of OSAS mode IH on pulmonary fibrosis induced by bleomycin in mice. Effect of OSAS mode IH on activation and extracellular matrix secretion of lung fibroblasts in mice. Content 1.OSAS model IH affects pulmonary fibrosis induced by bleomycin in mice. The effect of.2.OSAS mode IH on the activation of lung fibroblasts and extracellular matrix secretion of mice is established by the method of establishing a model of bleomycin induced pulmonary fibrosis in mice. Then randomly divided into 3 groups, group N (Chang Yangzu), group IH (intermittent hypoxic group) and group IH+T (Tempol intervention group). For IH+T group, Tempol intraperitoneal injection was given half an hour before IH treatment every day. The 3 groups of mice were placed in the self-made IH exposure container, and the compressed air was continuously transported to the N group in the 10:00-16:00 period, to the IH group and the IH+T group. The mixture of oxygen and nitrogen gas, Western blot method was used to detect the alpha -SMA, and the cells of the cell culture box were cultured in the cell culture box to pass the cell line MLg cells. When the cells were long to 80-90% fusion, they were inoculated in the culture dish, and after the culture of 48h, the adjusted lung fibroblast MLg was randomly divided into 5 groups, and 4 groups were placed in the IH experimental cabin, and 1 groups were randomly selected. 8 hours before IH treatment, Tempol intervention was carried out, OSAS mode IH stimulation was given by computer program control device, 1H, 4h, 8h, and T+8h, the control group cells were placed in the incubator to cultivate 24h, and then the intermittent hypoxia group and the control group cells and the supernatant were collected respectively. The alpha -SMA was detected by Q RT-PCR method and the expression was detected by Q RT-PCR method. SMA, type I collagen (COL1) protein expression. Another statistical analysis used SPSS16.0 software package, and the data were expressed as x + s. Multiple groups were compared with single factor analysis of variance (One-Way ANOVA), P0.05, difference was significant, P0.01, and the difference was very significant. Effect of level and effect of Tempol intervention on the expression level of alpha -SMA protein, Western blot analysis showed that the expression level of alpha -SMA protein IH group in lung tissue of mice induced by bleomycin was significantly higher than that of normal oxygen control group, the difference was statistically significant (P0.05), and the expression level of alpha -SMA protein in IH+T group was more than that of the IH group. Lower, but higher than the normal oxygen control group, the difference has statistical significance (P0.05).2. in different intermittent hypoxia time, the effect of alpha -SMA expression in MLg fibroblasts and the effect of Tempol intervention on the expression of alpha -SMA protein: the Q RT-PCR results show that IH1h, IH4h, IH8h mouse lung MLg fibroblasts are relative water The relative expression level of alpha -SMA m RNA in lung MLg cells in IH1h and IH4h mice increased significantly (P0.05), and the expression level of alpha -SMA m RNA in the lung MLg cells of IH8h mice increased with the prolongation of IH time (F=104.3 P0.05). The expression level of alpha -SMA protein in lung MLg fibroblasts in IH1h, IH4h, IH8h mice increased with the prolongation of IH time (P0.05), and the expression level of.IH8h+T alpha -SMA protein in IH8h (P0.05) was higher than that in IH8h (P0.05). The effect of the expression level of COL1 and the effect of Tempol intervention on the expression of COL1 protein: Q RT-PCR results showed that the relative expression level of COL1 m RNA in IH1h, IH4h, IH8h mice lung MLg fibroblasts was significantly higher than that in the normal oxygen control group. =220.3 P0.05), and the peak (P0.01).Western blot analysis in IH8h group showed that the expression level of COL1 protein in IH1h, IH4h, IH8h mouse lung MLg fibroblasts was higher than that of the normal oxygen control group (P0.05). The expression level of.IH8h+T COL1 protein protein was higher than that of IH1h, IH4h, IH8h, but slightly higher than that of group N (P0.01). Conclusion 1. in OSA mode IH can aggravate the pulmonary fibrosis induced by bleomycin, and the intervention of antioxidant Tempol can weaken the promotion of pulmonary fibrosis induced by bloomicamycin in OSAS mode. Model IH can promote pulmonary fibroblast activation and extracellular matrix secretion 3. antioxidant Tempol intervention to reduce the promotion of IH to pulmonary fibroblast activation and extracellular matrix secretion in OSAS mode, and the 4. oxidative stress response plays an important role in the process of OSA mode IH to promote pulmonary fibrosis.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R766
本文編號：2174744
[Abstract]:Obstructive sleep apnea syndrome (OSAS) is a chronic sleep respiratory disease, which is characterized by repeated upper airway or complete obstruction of the upper airway during sleep, with a prevalence rate of 2%~ 4% in adults and a 5 year mortality of 11%~13% in untreated OSAS patients. Intermittent hypoxia (IH) is a unique pathophysiological change of OSAS, which is characterized by hypoxic reoxygenation, similar to ischemia / reperfusion injury, and is considered to be associated with a variety of OSAS complications such as cardiovascular and cerebrovascular diseases, metabolic disorders, cognitive dysfunction, and genitourinary dysfunction. In recent years, the relationship between OSAS and pulmonary fibrosis has been gradually discovered by researchers. Pulmonary fibrosis is a progressive, fatal, interstitial lung disease with unknown etiology, which is a large accumulation of fibroblast / myofibroblast (fibroblasts/myofibroblasts). The average survival time of pulmonary fibrosis was 2 years after diagnosis, the average survival rate of pulmonary fibrosis was 2 years, the 5 year survival rate was 30%~50%, the mortality rate was high, and the prognosis was poor. At present, the clinical lack of effective treatment,.OSAS is a systemic disease, it can merge a variety of diseases, and oxidative stress is OSAS. The common mechanism of pulmonary fibrosis. This study will preliminarily discuss the effect of OSAS model IH on pulmonary fibrosis and discuss the effect of oxidative stress on it, and provide experimental basis and theoretical basis for further clinical study. Objective 1 to explore the effect of OSAS mode IH on pulmonary fibrosis induced by bleomycin in mice. Effect of OSAS mode IH on activation and extracellular matrix secretion of lung fibroblasts in mice. Content 1.OSAS model IH affects pulmonary fibrosis induced by bleomycin in mice. The effect of.2.OSAS mode IH on the activation of lung fibroblasts and extracellular matrix secretion of mice is established by the method of establishing a model of bleomycin induced pulmonary fibrosis in mice. Then randomly divided into 3 groups, group N (Chang Yangzu), group IH (intermittent hypoxic group) and group IH+T (Tempol intervention group). For IH+T group, Tempol intraperitoneal injection was given half an hour before IH treatment every day. The 3 groups of mice were placed in the self-made IH exposure container, and the compressed air was continuously transported to the N group in the 10:00-16:00 period, to the IH group and the IH+T group. The mixture of oxygen and nitrogen gas, Western blot method was used to detect the alpha -SMA, and the cells of the cell culture box were cultured in the cell culture box to pass the cell line MLg cells. When the cells were long to 80-90% fusion, they were inoculated in the culture dish, and after the culture of 48h, the adjusted lung fibroblast MLg was randomly divided into 5 groups, and 4 groups were placed in the IH experimental cabin, and 1 groups were randomly selected. 8 hours before IH treatment, Tempol intervention was carried out, OSAS mode IH stimulation was given by computer program control device, 1H, 4h, 8h, and T+8h, the control group cells were placed in the incubator to cultivate 24h, and then the intermittent hypoxia group and the control group cells and the supernatant were collected respectively. The alpha -SMA was detected by Q RT-PCR method and the expression was detected by Q RT-PCR method. SMA, type I collagen (COL1) protein expression. Another statistical analysis used SPSS16.0 software package, and the data were expressed as x + s. Multiple groups were compared with single factor analysis of variance (One-Way ANOVA), P0.05, difference was significant, P0.01, and the difference was very significant. Effect of level and effect of Tempol intervention on the expression level of alpha -SMA protein, Western blot analysis showed that the expression level of alpha -SMA protein IH group in lung tissue of mice induced by bleomycin was significantly higher than that of normal oxygen control group, the difference was statistically significant (P0.05), and the expression level of alpha -SMA protein in IH+T group was more than that of the IH group. Lower, but higher than the normal oxygen control group, the difference has statistical significance (P0.05).2. in different intermittent hypoxia time, the effect of alpha -SMA expression in MLg fibroblasts and the effect of Tempol intervention on the expression of alpha -SMA protein: the Q RT-PCR results show that IH1h, IH4h, IH8h mouse lung MLg fibroblasts are relative water The relative expression level of alpha -SMA m RNA in lung MLg cells in IH1h and IH4h mice increased significantly (P0.05), and the expression level of alpha -SMA m RNA in the lung MLg cells of IH8h mice increased with the prolongation of IH time (F=104.3 P0.05). The expression level of alpha -SMA protein in lung MLg fibroblasts in IH1h, IH4h, IH8h mice increased with the prolongation of IH time (P0.05), and the expression level of.IH8h+T alpha -SMA protein in IH8h (P0.05) was higher than that in IH8h (P0.05). The effect of the expression level of COL1 and the effect of Tempol intervention on the expression of COL1 protein: Q RT-PCR results showed that the relative expression level of COL1 m RNA in IH1h, IH4h, IH8h mice lung MLg fibroblasts was significantly higher than that in the normal oxygen control group. =220.3 P0.05), and the peak (P0.01).Western blot analysis in IH8h group showed that the expression level of COL1 protein in IH1h, IH4h, IH8h mouse lung MLg fibroblasts was higher than that of the normal oxygen control group (P0.05). The expression level of.IH8h+T COL1 protein protein was higher than that of IH1h, IH4h, IH8h, but slightly higher than that of group N (P0.01). Conclusion 1. in OSA mode IH can aggravate the pulmonary fibrosis induced by bleomycin, and the intervention of antioxidant Tempol can weaken the promotion of pulmonary fibrosis induced by bloomicamycin in OSAS mode. Model IH can promote pulmonary fibroblast activation and extracellular matrix secretion 3. antioxidant Tempol intervention to reduce the promotion of IH to pulmonary fibroblast activation and extracellular matrix secretion in OSAS mode, and the 4. oxidative stress response plays an important role in the process of OSA mode IH to promote pulmonary fibrosis.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R766
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