CXCR4在肺型氧中毒致肺损伤机制中的作用研究
发布时间：2018-07-25 13:34
【摘要】：在潜水作业及高压氧治疗中,吸入气氧分压过高或吸氧时间过长导致肺部的功能和结构发生病理性变化而表现出的症状称之为肺型氧中毒。肺型氧中毒出现的症状易被脑型氧中毒的神经系统症状掩盖,且发病机制尚未完全明确,因此切实有效的干预措施和治疗手段并不完善。若不及时进行治疗,一旦进展为急性肺损伤甚至呼吸窘迫综合症,患者就可能进入全身多器官衰竭状态,预后极差。肺型氧中毒发生时肺内肺泡-毛细血管屏障遭到破坏、出现气体交换障碍、肺内发生炎症反应和肺水肿等病理学改变。在炎性因子和趋化因子的共同作用下,大量炎症细胞在肺内募集和黏附,不仅破坏肺脏血管内皮屏障,还同时释放大量的蛋白酶等物质,进一步加重肺内炎症反应程度和上皮细胞损伤,进而诱导更多信号转导通路和转录因子的激活,形成所谓“放大”效应。肺损伤的严重程度与炎症细胞浸润密切相关,而炎症细胞浸润程度则与其趋化能力有关。因此,肺型氧中毒发生时,降低炎症细胞的趋化能力和肺内炎症反应以及调节相关信号转导通路活性成为目前治疗氧中毒肺损伤的主要思路。趋化因子受体4(CXC Chemokine Receptor 4,CXCR4)属于G蛋白耦联受体蛋白超家族的一员,在炎症反应、病原体清除等方面起到调节信号转导、趋化炎症细胞、维持炎性反应动态平衡等重要作用。相关研究发现,高压氧处理后大鼠肺组织出现出血、水肿,同时CXCR4表达显著升高。据此推测其可能的机制为高压氧状态下血管内皮细胞、肺泡上皮细胞损伤后,升高的CXCR4通过激活G蛋白耦联的信号通路及转录因子引起细胞损伤、凋亡并释放一系列炎症因子,进一步损伤肺组织。但是其具体的损伤机制如相关信号转导通路、下游炎症因子水平变化及CXCR4在其中的作用机制仍不十分清楚。由此,本课题进行了以下研究:1、肺型氧中毒致肺损伤中CXCR4表达的变化规律:通过0.23MPa纯氧高压暴露复制动物模型,观察高压氧作用下不同暴露时间大鼠肺组织病理学改变,利用肺湿干重比、BALF总蛋白含量来判断肺通透性变化,应用Western Blot方法研究肺组织CXCR4表达变化,ELISA法检测炎症因子TNF-α、IL-1β作为衡量下游炎症因子水平变化依据,探索肺损伤随高压氧暴露不同时间的变化规律,并对湿干重比、BALF总蛋白、TNF-α、IL-1β及CXCR4指标变化与UPTD剂量值进行相关性分析以探讨CXCR4随肺损伤程度变化而变化的规律及机制。2、CXCR4抑制剂对肺型氧中毒致肺损伤的干预作用:根据前期实验结果,选择指标变化最显著的0.23MPa纯氧暴露8小时方案,利用CXCR4特异性阻断剂AMD3100进行预处理,并从大鼠肺组织病理学改变、肺通透性变化及CXCR4表达变化角度判断AMD3100对大鼠肺型氧中毒致肺损伤的干预作用,观察检测炎症因子TNF-α、IL-1β及凋亡蛋白Caspase3含量变化以研究下游信号通路在抑制剂干预下的变化情况,探究肺损伤中CXCR4抑制剂对肺型氧中毒致肺损伤的干预作用机制,进一步验证CXCR4在肺型氧中毒致肺损伤机制中的作用。主要研究结果如下:1、本工作成功复制了0.23MPa高压氧暴露不同时程大鼠肺型氧中毒模型,为下一步实验奠定了实验基础。2、随着高压氧暴露时间的延长,大鼠肺组织病理学变化、肺通透性逐渐加重,与肺组织损伤程度相一致;高压氧暴露后肺组织CXCR4表达明显升高(P0.01),随暴露时间变化逐渐升高,并在8小时组达到高峰,与炎症因子TNF-α含量、IL-1β含量具有相同的变化趋势。不同高压氧暴露时间大鼠肺组织通透性指标、肺组织CXCR4、TNF-α及IL-1β含量与UPTD剂量均呈正相关(P0.01)。3、常压空气条件下预先应用CXCR4特异性抑制剂AMD3100处理大鼠,与使用PBS预处理相比,肺组织中CXCR4、TNF-α、IL-1β及活化的Caspase-3含量变化差异无显著性(P0.05);而在高压氧暴露下预先应用AMD3100处理与PBS预处理相比,大鼠肺组织中CXCR4、TNF-α、IL-1β及活化的Caspase-3含量降低,差异有显著性(P0.01)。同时,应用AMD3100预处理后大鼠肺组织病理学检查及通透性指标也有一定程度的减轻。表明CXCR4特异性抑制剂AMD3100可减轻大鼠肺型氧中毒致肺损伤程度,靶向CXCR4的肺型氧中毒致肺损伤治疗方案更具针对性和选择性,值得进行深入研究。综上所述,本课题阐明了CXCR4在肺型氧中毒致肺损伤中发挥作用的可能机制,为找到干预肺型氧中毒致肺损伤有效靶点提供了新的依据,为潜水作业及临床治疗提供有效的医学保障。
[Abstract]:In the submersible operation and hyperbaric oxygen therapy, the symptoms of pulmonary oxygen poisoning are called pulmonary oxygen poisoning. The symptoms of pulmonary oxygen poisoning are easily concealed by the nervous system symptoms of brain oxygen poisoning, and the pathogenesis is not completely clear, so the pathogenesis is not completely clear. The effective intervention measures and treatment methods are not perfect. If they are not treated in time, the patients may enter the state of multiple organ failure, and the prognosis is extremely poor. With the joint action of inflammatory and chemokines, a large number of inflammatory cells collect and adhere to the lungs, not only destroy the blood vessel endothelial barrier, but also release a large number of proteases, which further aggravate the degree of inflammation of the lungs and the injury of epithelial cells. The activation of multiple signal transduction pathways and transcription factors form the so-called "amplification" effect. The severity of the lung injury is closely related to the infiltration of inflammatory cells, and the degree of infiltration of inflammatory cells is related to the chemotaxis. Therefore, when pulmonary oxygen poisoning occurs, it reduces the chemotaxis and inflammatory responses of the inflammatory cells and regulates the related letters. The activity of signal transduction pathway is the main idea for the treatment of lung injury in oxygen poisoning. Chemokine receptor 4 (CXC Chemokine Receptor 4, CXCR4) is a member of the protein superfamily of G protein coupled receptor. It plays an important role in regulating signal transduction, chemotaxis of inflammatory cells and maintaining dynamic balance of inflammatory reaction in the aspects of inflammatory reaction and pathogen clearance. It was found that after hyperbaric oxygen treatment, the lung tissue appeared bleeding, edema, and the expression of CXCR4 increased significantly. Accordingly, it was suggested that the possible mechanism was vascular endothelial cells in hyperbaric oxygen state. After the alveolar epithelial cells were damaged, the elevated CXCR4 caused cell damage by activating the signal pathway and transcription factors of G protein coupling and withering. A series of inflammatory factors are released to further damage the lung tissue, but the specific damage mechanisms such as related signal transduction pathways, the level of downstream inflammatory factors and the mechanism of CXCR4 in it are still not very clear. Therefore, the following studies have been carried out: 1, the changes in the expression of CXCR4 in lung injury induced by pulmonary oxygen poisoning: Over 0.23MPa pure oxygen high pressure exposure animal model, observe the pathological changes of lung tissue in rats with different exposure time under hyperbaric oxygen, use the ratio of wet dry weight of lung, BALF total protein content to determine lung permeability change, use Western Blot method to study the change of CXCR4 expression in lung tissue, ELISA method to detect the inflammatory factor TNF- a, IL-1 beta as the measure The change of lung injury with hyperbaric oxygen exposure at different time, and the correlation analysis of the changes of wet dry weight ratio, BALF total protein, TNF- alpha, IL-1 beta and CXCR4 and UPTD dose value to explore the regularities and mechanism of CXCR4 with the change of lung injury degree and mechanism.2, CXCR4 inhibitor in lung oxygen oxygen. The intervention effect of toxic lung injury: according to the previous experimental results, the most significant 0.23MPa oxygen exposure scheme was selected for 8 hours, and the CXCR4 specific blocker AMD3100 was pretreated. The lung tissue pathological changes, pulmonary permeability change and CXCR4 expression change angle were used to determine the lung damage induced by AMD3100 on lung oxygen poisoning in rats. The changes of the inflammatory factors TNF- alpha, IL-1 beta and apoptotic protein Caspase3 were observed and measured to study the changes in the downstream signal pathway under the intervention of inhibitors, and to explore the mechanism of intervention of CXCR4 inhibitors on lung injury induced by pulmonary oxygen poisoning in lung injury, and to further verify the mechanism of CXCR4 in lung injury induced lung injury mechanism. The main results are as follows: 1, this work successfully replicates the lung oxygen poisoning model of 0.23MPa hyperbaric oxygen exposure in different time course rats and lays a foundation for the next experiment. With the prolonged exposure of hyperbaric oxygen, the pathological changes of lung tissue and lung permeability are gradually aggravated, and the degree of lung injury is consistent with the degree of lung tissue injury. The expression of CXCR4 in lung tissue increased significantly (P0.01) after exposure to oxygen exposure, and increased gradually with the exposure time, and reached the peak in the 8 hour group. The content of TNF- alpha and inflammatory factor, IL-1 beta content had the same trend. The lung tissue permeability index of rats with different hyperbaric oxygen exposure time, the content of CXCR4, TNF- A and IL-1 beta in the lung tissue were all positive with the dose of UPTD. There was no significant difference in CXCR4, TNF- a, IL-1 beta and activation of CXCR4, TNF- a, IL-1 beta and activated Caspase-3 (P0.05) in lung tissue compared with the use of CXCR4 specific inhibitor AMD3100 under normal pressure air conditions, compared with PBS preconditioning (P0.05), while in hyperbaric oxygen exposure, AMD3100 treatment was compared with PBS preconditioning in the lung tissue of rats. CXCR4, TNF- alpha, IL-1 beta and activated Caspase-3 content decreased significantly (P0.01). At the same time, the pathological examination and permeability index of lung tissue of rats were also reduced to a certain extent after AMD3100 pretreatment. It showed that CXCR4 specific inhibitor AMD3100 could reduce lung injury induced by pulmonary oxygen poisoning in rats and target CXCR4 in lung oxygen oxygen. The treatment scheme for lung injury is more targeted and selective and deserves in-depth study. To sum up, this topic illustrates the possible mechanism of the role of CXCR4 in lung injury induced by pulmonary oxygen poisoning. It provides a new basis for finding effective targets for the intervention of lung injury caused by pulmonary oxygen poisoning and provides effective treatment for submersible operation and clinical treatment. Medical security.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R595
本文编号：2143982
[Abstract]:In the submersible operation and hyperbaric oxygen therapy, the symptoms of pulmonary oxygen poisoning are called pulmonary oxygen poisoning. The symptoms of pulmonary oxygen poisoning are easily concealed by the nervous system symptoms of brain oxygen poisoning, and the pathogenesis is not completely clear, so the pathogenesis is not completely clear. The effective intervention measures and treatment methods are not perfect. If they are not treated in time, the patients may enter the state of multiple organ failure, and the prognosis is extremely poor. With the joint action of inflammatory and chemokines, a large number of inflammatory cells collect and adhere to the lungs, not only destroy the blood vessel endothelial barrier, but also release a large number of proteases, which further aggravate the degree of inflammation of the lungs and the injury of epithelial cells. The activation of multiple signal transduction pathways and transcription factors form the so-called "amplification" effect. The severity of the lung injury is closely related to the infiltration of inflammatory cells, and the degree of infiltration of inflammatory cells is related to the chemotaxis. Therefore, when pulmonary oxygen poisoning occurs, it reduces the chemotaxis and inflammatory responses of the inflammatory cells and regulates the related letters. The activity of signal transduction pathway is the main idea for the treatment of lung injury in oxygen poisoning. Chemokine receptor 4 (CXC Chemokine Receptor 4, CXCR4) is a member of the protein superfamily of G protein coupled receptor. It plays an important role in regulating signal transduction, chemotaxis of inflammatory cells and maintaining dynamic balance of inflammatory reaction in the aspects of inflammatory reaction and pathogen clearance. It was found that after hyperbaric oxygen treatment, the lung tissue appeared bleeding, edema, and the expression of CXCR4 increased significantly. Accordingly, it was suggested that the possible mechanism was vascular endothelial cells in hyperbaric oxygen state. After the alveolar epithelial cells were damaged, the elevated CXCR4 caused cell damage by activating the signal pathway and transcription factors of G protein coupling and withering. A series of inflammatory factors are released to further damage the lung tissue, but the specific damage mechanisms such as related signal transduction pathways, the level of downstream inflammatory factors and the mechanism of CXCR4 in it are still not very clear. Therefore, the following studies have been carried out: 1, the changes in the expression of CXCR4 in lung injury induced by pulmonary oxygen poisoning: Over 0.23MPa pure oxygen high pressure exposure animal model, observe the pathological changes of lung tissue in rats with different exposure time under hyperbaric oxygen, use the ratio of wet dry weight of lung, BALF total protein content to determine lung permeability change, use Western Blot method to study the change of CXCR4 expression in lung tissue, ELISA method to detect the inflammatory factor TNF- a, IL-1 beta as the measure The change of lung injury with hyperbaric oxygen exposure at different time, and the correlation analysis of the changes of wet dry weight ratio, BALF total protein, TNF- alpha, IL-1 beta and CXCR4 and UPTD dose value to explore the regularities and mechanism of CXCR4 with the change of lung injury degree and mechanism.2, CXCR4 inhibitor in lung oxygen oxygen. The intervention effect of toxic lung injury: according to the previous experimental results, the most significant 0.23MPa oxygen exposure scheme was selected for 8 hours, and the CXCR4 specific blocker AMD3100 was pretreated. The lung tissue pathological changes, pulmonary permeability change and CXCR4 expression change angle were used to determine the lung damage induced by AMD3100 on lung oxygen poisoning in rats. The changes of the inflammatory factors TNF- alpha, IL-1 beta and apoptotic protein Caspase3 were observed and measured to study the changes in the downstream signal pathway under the intervention of inhibitors, and to explore the mechanism of intervention of CXCR4 inhibitors on lung injury induced by pulmonary oxygen poisoning in lung injury, and to further verify the mechanism of CXCR4 in lung injury induced lung injury mechanism. The main results are as follows: 1, this work successfully replicates the lung oxygen poisoning model of 0.23MPa hyperbaric oxygen exposure in different time course rats and lays a foundation for the next experiment. With the prolonged exposure of hyperbaric oxygen, the pathological changes of lung tissue and lung permeability are gradually aggravated, and the degree of lung injury is consistent with the degree of lung tissue injury. The expression of CXCR4 in lung tissue increased significantly (P0.01) after exposure to oxygen exposure, and increased gradually with the exposure time, and reached the peak in the 8 hour group. The content of TNF- alpha and inflammatory factor, IL-1 beta content had the same trend. The lung tissue permeability index of rats with different hyperbaric oxygen exposure time, the content of CXCR4, TNF- A and IL-1 beta in the lung tissue were all positive with the dose of UPTD. There was no significant difference in CXCR4, TNF- a, IL-1 beta and activation of CXCR4, TNF- a, IL-1 beta and activated Caspase-3 (P0.05) in lung tissue compared with the use of CXCR4 specific inhibitor AMD3100 under normal pressure air conditions, compared with PBS preconditioning (P0.05), while in hyperbaric oxygen exposure, AMD3100 treatment was compared with PBS preconditioning in the lung tissue of rats. CXCR4, TNF- alpha, IL-1 beta and activated Caspase-3 content decreased significantly (P0.01). At the same time, the pathological examination and permeability index of lung tissue of rats were also reduced to a certain extent after AMD3100 pretreatment. It showed that CXCR4 specific inhibitor AMD3100 could reduce lung injury induced by pulmonary oxygen poisoning in rats and target CXCR4 in lung oxygen oxygen. The treatment scheme for lung injury is more targeted and selective and deserves in-depth study. To sum up, this topic illustrates the possible mechanism of the role of CXCR4 in lung injury induced by pulmonary oxygen poisoning. It provides a new basis for finding effective targets for the intervention of lung injury caused by pulmonary oxygen poisoning and provides effective treatment for submersible operation and clinical treatment. Medical security.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R595
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