OSAS模式IH对小鼠肺纤维化的影响及其机制的初步研究
发布时间：2018-08-09 17:06
【摘要】：阻塞性睡眠呼吸暂停综合征(obstructive sleep apnea syndrome,OSAS)是一种以睡眠过程中反复发生上气道部分或者完全阻塞为特点导致睡眠结构紊乱的慢性睡眠呼吸系统疾病,其在成年人的患病率为2%~4%,未经治疗的OSAS患者5年病死率达11%~13%,严重威胁着患者的健康和生命。间歇低氧(intermittent hypoxia,IH)是OSAS独特的病理生理学改变,其特点为低氧-复氧间断发生,类似于缺血/再灌注损伤,被认为与OSAS多种并发症如:心脑血管疾病、代谢紊乱疾病、认知功能障碍、生殖泌尿功能异常和癌症等有关,对机体的危害是不容小觑的。近几年,OSAS与肺间质纤维化的联系逐渐被研究人员发现,肺间质纤维化是一种进行性发展、致死性的、病因不明的间质性肺疾病,它是由纤维母细胞/肌纤母细胞(fibroblasts/myofibroblasts)大量聚集和胶原(collagen)等胞外基质(extracellular matrix,ECM)过度沉积造成的。肺纤维化确诊后平均存活期为2年,5年生存率为30%~50%,病死率高,预后差,目前临床缺乏有效治疗方法。OSAS是一种系统性的疾病,它可以合并多种疾病,而氧化应激是OSAS与肺间质纤维化的共同作用机制。本研究将要初步探讨OSAS模式IH对肺纤维化的影响,并讨论氧化应激对在其中所产生的的影响,为进一步临床研究提供实验基础和理论依据。研究目的1、探讨OSAS模式IH对博来霉素诱导的小鼠肺纤维化的影响。2、探讨OSAS模式IH对小鼠肺成纤维细胞活化和胞外基质分泌的影响。内容1.OSAS模式IH对博来霉素诱导的小鼠肺纤维化的影响。2.OSAS模式IH对小鼠肺成纤维细胞活化和胞外基质分泌的影响方法建立博来霉素诱导的小鼠肺纤维化的模型,后随机分成3组,N组(常氧组),IH组(间歇低氧组)以及IH+T组(Tempol干预组)。对于IH+T组,每天于IH处理前半小时予Tempol腹腔注射,将3组小鼠分别放置在自制IH暴露容器内,在10:00-16:00的时段内分别向N组持续输送压缩空气,向IH组和IH+T组间断输送氧气和氮气的混合气体,Western blot法检测α-SMA,另于细胞培养箱内培养成纤维细胞可传代细胞株MLg细胞,待细胞长至80-90%融合时,接种于培养皿内,培养48h后,将调整好的肺成纤维细胞MLg随机分成5组,将4组置于IH实验舱内,随机选择1组在IH处理8小时前,进行Tempol的干预,通过计算机程控装置分别给予OSAS模式IH刺激,1h、4h、8h,及T+8h,将对照组细胞放置在培养箱内培养24h,然后分别收集间歇低氧组和对照组细胞及上清,通过q RT-PCR法检测α-SMA,Col1a1的表达;Western blot法检测α-SMA,type I collagen(COL1)蛋白的表达。另统计分析采用SPSS16.0软件包,数据以x±s表示.多组间比较采用单因素方差分析(One-Way ANOVA),P0.05,差异显著,P0.01,差异极显著。结果1.OSAS模式IH对博来霉素诱导的小鼠肺纤维化模型中肺组织α-SMA蛋白表达水平的影响及Tempol干预对α-SMA蛋白表达水平的影响Western blot分析结果表明:在博来霉素诱导的小鼠肺纤维化模型中肺组织α-SMA蛋白IH组的表达水平较常氧对照组明显增高,差异具备统计学意义(P0.05),IH+T组α-SMA蛋白的表达水平较IH组降低,但高于常氧对照组,差异具备统计学意义(P0.05)。2.不同间歇低氧时间下肺MLg成纤维细胞中α-SMA表达水平的影响以及Tempol干预对α-SMA蛋白表达水平的影响:q RT-PCR结果表明:IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞中α-SMA m RNA的相对表达水平较常氧对照组明显增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg细胞中α-SMA m RNA的相对表达水平随IH时间延长而表达增多(F=104.3 P0.05),并且在IH8h达到高峰(P0.01)。Western blot分析结果表明:IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞中α-SMA蛋白的表达水平较常氧对照组增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞α-SMA蛋白的表达水平随IH时间延长而表达增多(P0.05),并且在IH8h达到高峰(P0.05)。IH8h+Tα-SMA蛋白的表达水平较IH1h,IH4h,IH8h降低,也较N组降低(P0.01)。3.不同间歇低氧时间下肺MLg成纤维细胞中COL1表达水平的影响以及Tempol干预对COL1蛋白表达水平的影响:q RT-PCR结果表明:IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞中COL1 m RNA相对表达水平较常氧对照组明显增高(P0.05),IH1h,IH4h,IH8h细胞中COL1m RNA的相对表达水平随IH时间延长而表达增多(F=220.3 P0.05),并且在IH8h组达到高峰(P0.01)。Western blot分析结果表明:IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞COL1蛋白的表达水平较常氧对照组增高(P0.05),IH1h,IH4h,IH8h小鼠肺MLg成纤维细胞COL1蛋白的表达水平随IH时间延长而表达增多(P0.05),并且在IH8h达到高峰P0.05)。IH8h+T COL1蛋白蛋白的表达水平较IH1h,IH4h,IH8h降低,但稍高于N组(P0.01)。结论1.在OSA模式IH可以加重博来霉素诱导的小鼠肺纤维化;抗氧化剂Tempol的干预可以减弱OSAS模式下IH对博来霉素诱导的小鼠肺纤维化的促进作用。2.OSAS模式IH可以促进肺成纤维细胞活化和胞外基质分泌3.抗氧化剂Tempol的干预可以减弱OSAS模式下IH对肺成纤维细胞活化和跑外基质分泌的促进作用;4.氧化应激反应在OSA模式IH促进肺纤维化的过程中有重要的调节作用。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766
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