自噬在肺缺血再灌注损伤中的作用及其机制研究
发布时间：2019-06-03 08:17
【摘要】：背景：肺缺血再灌注损伤是肺移植、袖式肺叶手术、心脏外科手术、心肺复苏后综合征等疾病中常见的引起不良后果的病理生理过程。以肺移植为例,有研究证实,急慢性肺移植排斥反应的发生均与肺移植术后早期肺缺血再灌注损伤的程度有关。已有的研究表明,肺缺血再灌注损伤主要表现为肺间质水肿、炎症细胞浸润、呼吸膜断裂和气体交换障碍。然而,肺缺血再灌注损伤发生发展的整体机制仍未被全面了解。 自噬是真核生物进化高度保守的细胞自稳程序。一方面,双膜结构的自噬囊泡能包绕隔绝失功能的细胞器、结构破坏的蛋白质和侵入细胞的病原体等。另一方面,自噬体与溶酶体结合消化囊内物进而为细胞代谢提供底物。生理状态下细胞内一定水平的自噬对维持细胞内环境的稳定起重要作用。当被过度诱导时,自噬也能导致细胞程序性死亡,与凋亡不同,这一过程被称之为Ⅱ型程序性死亡。另外,由于自噬与凋亡的复杂联系,一定程度的自噬甚至直接诱导细胞凋亡。因此,细胞自噬被认为是细胞在压力条件下的适应机制。 国内外越来越多的研究发现自噬在许多病理生理学状态中扮演重要作用,例如肿瘤、感染、神经退行性疾病等。国外有研究证实缺血再灌注过程能够诱导自噬水平的快速上调。Chigure Suzuki等人的研究认为自噬参与了肾脏的缺血再灌注损伤。然而,Man Jiang等在另一个肾脏缺血再灌注损伤模型中发现自噬在肾脏缺血再灌注损伤中是一种肾脏保护因素。在肝脏中,Kunihito Gotoh等发现药物抑制自噬能够改善肝移植术后的肝脏缺血再灌注损伤程度。因此,有学者推测自噬在缺血再灌注损伤中的作用可能是组织和模型依耐性的。当前,自噬较少涉及呼吸系统疾病的研究,仅在吸烟、COPD、肺动脉高压等慢性呼吸系统疾病的肺组织中发现自噬水平升高,高水平的自噬与细胞的凋亡,组织纤维化与重构密切相关。许多学者预测：自噬将成为肺部疾病基础研究中一个新的前沿领域。那么,肺缺血再灌注时肺内细胞自噬水平如何?自噬在肺缺血再灌注损伤中作用如何?本研究试图在大鼠左肺原位热缺血再灌注损伤模型中寻找答案。 目的：建立大鼠左肺原位热缺血再灌注损伤模型,观测大鼠肺缺血再灌注后肺组织内细胞自噬水平的变化,探讨肺缺血及再灌注对细胞自噬流的影响。药物干预自噬,研究自噬在肺缺血再灌注损伤中的作用,并初步探索自噬在肺缺血再灌注损伤中发挥作用的可能机制。 方法：以SPF级SD大鼠作为实验动物,建立左肺原位热缺血再灌注损伤模型。运用免疫印迹法检测缺血1h和再灌注0、2、6、24h后肺组织内自噬标志蛋白LC3表达水平,免疫荧光染色法双标自噬体标志蛋白LC3与溶酶体膜蛋白LAMP2,鉴定自噬体与溶酶体融合降解是否正常,电子显微镜观测细胞内自噬体,分析大鼠左肺缺血再灌注后肺组织内细胞自噬水平变化情况。比较缺血3h、缺血1h再灌注2h、阳性对照组、假手术组自噬水平,分析缺血和再灌注对自噬的诱导作用。自噬抑制剂3-甲基腺嘌呤(3-MA)和安慰剂分别预处理大鼠,检测左肺缺血1h再灌注2h后肺干湿重比(Lung W/DRatio),MDA (malondialdehyde)浓度,MPO (myeloperoxidase)活性等肺损伤指标,HE染色评价肺组织病理学损伤程度,并验证3-MA抑制细胞自噬能力,分析自噬在肺缺血再灌注损伤中的作用。免疫印迹法检测3-MA处理组和对照组肺组织中凋亡关键蛋白cleaved caspas-3表达水平,TUNEL (terminal dUTP nick end-labeling assay)法检测肺组织中凋亡细胞比率,分析自噬参与肺缺血再灌注损伤的可能机制。 结果：在大鼠左肺热缺血再灌注损伤模型中,自噬水平在缺血1h即有升高(P0.05),再灌注2-6h达到高峰(P0.01),再灌组24h恢复至接近假手术组水平(P0.05),电子显微镜检查主要在Ⅰ型肺泡上皮细胞和血管内皮细胞内观察到自噬体。缺血1h再灌注2h较缺血3h自噬水平高,与雷帕霉素处理的阳性对照组自噬水平相当。30mg/kg3-MA预处理与对照组比较降低肺组织损伤评分(P0.05),降低肺W/D,降低肺组织中MDA浓度和MPO活性。Western Blot结果示30mg/kg3-MA能有效抑制细胞自噬。同时,与对照组比较,3-MA预处理降低肺组织中cleaved caspas-3蛋白水平,降低凋亡细胞比率。 结论：肺缺血和再灌注过程均显著诱导肺组织细胞自噬水平上调。自噬药物性抑制剂3-MA预处理能够减轻肺缺血再灌注损伤程度,说明自噬在肺缺血再灌注中有加重损伤的作用。其机制可能与高水平的细胞自噬诱导细胞凋亡,损伤肺呼吸膜有关。
[Abstract]:BACKGROUND: The lung ischemia-reperfusion injury is a pathophysiological process of lung transplantation, sleeve-type lung-leaf operation, cardiac surgery, and post-pulmonary resuscitation syndrome. Taking lung transplantation as an example, it is proved that the occurrence of acute and chronic pulmonary graft rejection is related to the degree of early lung ischemia-reperfusion injury after lung transplantation. The existing studies have shown that lung ischemia-reperfusion injury is mainly characterized by pulmonary interstitial edema, inflammatory cell infiltration, respiratory membrane rupture and gas exchange disorder. However, the overall mechanism of the development of lung ischemia-reperfusion injury is still not fully understood. autophagy is a highly conserved, self-stable cell of eukaryote Order. On the one hand, the autophagy of the double membrane structure can be wrapped around the cell organelles that isolate the function of the protein, the proteins that are destroyed by the structure, and the pathogens that invade the cells. And the like. On the other hand, the self-phagemid and the lysosome combine to digest the capsule, thereby providing a bottom for the cell metabolism. A certain level of autophagy in the cells in a physiological state plays an important role in maintaining the stability of the intracellular environment In case of overinduction, autophagy can lead to programmed cell death, which is different from apoptosis, which is called type II programmed death. In addition, due to the complex association of autophagy and apoptosis, a certain degree of autophagy and even direct induction of the cell. The cell autophagy is considered to be an adaptation of the cell under pressure conditions. The more and more studies at home and abroad have found that autophagy plays an important role in many pathophysiological conditions, such as tumors, infections, neurodegenerative diseases, Diseases, etc. There are studies abroad to confirm that the ischemia-reperfusion process can induce a rapid autophagy. The study found that autophagy is involved in the ischemia-reperfusion of the kidney. Note injury. However, in another renal ischemia-reperfusion injury model, Man Jiang et al. found that autophagy is a kind of kidney protection in the renal ischemia-reperfusion injury. Protective factors. In the liver, Kunihito Gotoh et al. found that the inhibition of autophagy can improve the liver ischemia-reperfusion injury after liver transplantation The degree of injury is, therefore, that the role of autophagy in the ischemia-reperfusion injury may be the tissue and model. The current, autophagy is less involved in the study of respiratory diseases, and only in the lung of chronic respiratory diseases such as smoking, COPD, and pulmonary hypertension, autophagy is found to be elevated, high levels of autophagy and cell apoptosis, tissue fibrosis and remodeling Many scholars predict that autophagy will be a new one in the study of lung disease in that field, in the case of ischemia-reperfusion of the lung, the in-lung cell autophagy What's the level? autophagy in the lung ischemia-reperfusion injury How to use this study to find a model for in-situ thermal ischemia-reperfusion injury in the left lung of rats Objective: To establish a model of in-situ thermal ischemia-reperfusion injury in the left lung of rats and to observe the changes of autophagy in the lung after ischemia-reperfusion in rats. The effect of autophagy on the lung ischemia-reperfusion injury and the role of autophagy in the ischemia-reperfusion injury of the lung. The possible mechanism of left lung in situ thermal ischemia was established with SPF SD rats as experimental animals. The expression level of autophagy marker protein (LC3) in the lung tissue was detected by immunoblotting, and the expression level of the autophagy marker protein (LC3) in the lung tissue was detected by the immunoblotting method. The expression level of the autophagy marker protein (LC3) and the lysosomal membrane protein (LMP2) were identified by the immunofluorescent staining method. The self-phagocytosis and the lysosomal fusion were identified. Whether the in-situ degradation was normal or not, the self-phagocytosis of the cells was observed by the electron microscope, and the cell self-phagocytosis in the lung after the left lung ischemia and reperfusion in rats was analyzed. The changes of the level of autophagy were compared. The ischemia and reperfusion were compared with the control group and the sham-operation group for 2h, the positive control group and the sham-operation group. The induction of autophagy was induced by autophagy inhibitor 3-methyladenopterin (3-MA) and placebo, and the lung injury indexes such as lung and dry weight ratio (Lung W/ DRatio), MDA (malonic dehyde) concentration and MPO (myeloperoxidase) activity were detected after 1 h of left lung ischemia, and lung tissue was evaluated by HE staining. The degree of pathological injury, and the ability of 3-MA to inhibit autophagy, and the analysis of autophagy in the lung ischemia-reperfusion The apoptotic cell ratio in the lung tissue was detected by the terminal dUTP nick end-labeling assay in the 3-MA treatment group and the control group, and the autophagy was analyzed to participate in the lung ischemia-reperfusion. Results: In the model of ischemia-reperfusion injury in the left lung of rats, the level of autophagy increased (P0.05) in the ischemia-reperfusion injury model, then the peak was reached at 2-6 h (P0.01), and the reperfusion group was restored to the near-sham operation group for 24 h. The electron microscopic examination was mainly in type 鈪，
本文编号：2491792
[Abstract]:BACKGROUND: The lung ischemia-reperfusion injury is a pathophysiological process of lung transplantation, sleeve-type lung-leaf operation, cardiac surgery, and post-pulmonary resuscitation syndrome. Taking lung transplantation as an example, it is proved that the occurrence of acute and chronic pulmonary graft rejection is related to the degree of early lung ischemia-reperfusion injury after lung transplantation. The existing studies have shown that lung ischemia-reperfusion injury is mainly characterized by pulmonary interstitial edema, inflammatory cell infiltration, respiratory membrane rupture and gas exchange disorder. However, the overall mechanism of the development of lung ischemia-reperfusion injury is still not fully understood. autophagy is a highly conserved, self-stable cell of eukaryote Order. On the one hand, the autophagy of the double membrane structure can be wrapped around the cell organelles that isolate the function of the protein, the proteins that are destroyed by the structure, and the pathogens that invade the cells. And the like. On the other hand, the self-phagemid and the lysosome combine to digest the capsule, thereby providing a bottom for the cell metabolism. A certain level of autophagy in the cells in a physiological state plays an important role in maintaining the stability of the intracellular environment In case of overinduction, autophagy can lead to programmed cell death, which is different from apoptosis, which is called type II programmed death. In addition, due to the complex association of autophagy and apoptosis, a certain degree of autophagy and even direct induction of the cell. The cell autophagy is considered to be an adaptation of the cell under pressure conditions. The more and more studies at home and abroad have found that autophagy plays an important role in many pathophysiological conditions, such as tumors, infections, neurodegenerative diseases, Diseases, etc. There are studies abroad to confirm that the ischemia-reperfusion process can induce a rapid autophagy. The study found that autophagy is involved in the ischemia-reperfusion of the kidney. Note injury. However, in another renal ischemia-reperfusion injury model, Man Jiang et al. found that autophagy is a kind of kidney protection in the renal ischemia-reperfusion injury. Protective factors. In the liver, Kunihito Gotoh et al. found that the inhibition of autophagy can improve the liver ischemia-reperfusion injury after liver transplantation The degree of injury is, therefore, that the role of autophagy in the ischemia-reperfusion injury may be the tissue and model. The current, autophagy is less involved in the study of respiratory diseases, and only in the lung of chronic respiratory diseases such as smoking, COPD, and pulmonary hypertension, autophagy is found to be elevated, high levels of autophagy and cell apoptosis, tissue fibrosis and remodeling Many scholars predict that autophagy will be a new one in the study of lung disease in that field, in the case of ischemia-reperfusion of the lung, the in-lung cell autophagy What's the level? autophagy in the lung ischemia-reperfusion injury How to use this study to find a model for in-situ thermal ischemia-reperfusion injury in the left lung of rats Objective: To establish a model of in-situ thermal ischemia-reperfusion injury in the left lung of rats and to observe the changes of autophagy in the lung after ischemia-reperfusion in rats. The effect of autophagy on the lung ischemia-reperfusion injury and the role of autophagy in the ischemia-reperfusion injury of the lung. The possible mechanism of left lung in situ thermal ischemia was established with SPF SD rats as experimental animals. The expression level of autophagy marker protein (LC3) in the lung tissue was detected by immunoblotting, and the expression level of the autophagy marker protein (LC3) in the lung tissue was detected by the immunoblotting method. The expression level of the autophagy marker protein (LC3) and the lysosomal membrane protein (LMP2) were identified by the immunofluorescent staining method. The self-phagocytosis and the lysosomal fusion were identified. Whether the in-situ degradation was normal or not, the self-phagocytosis of the cells was observed by the electron microscope, and the cell self-phagocytosis in the lung after the left lung ischemia and reperfusion in rats was analyzed. The changes of the level of autophagy were compared. The ischemia and reperfusion were compared with the control group and the sham-operation group for 2h, the positive control group and the sham-operation group. The induction of autophagy was induced by autophagy inhibitor 3-methyladenopterin (3-MA) and placebo, and the lung injury indexes such as lung and dry weight ratio (Lung W/ DRatio), MDA (malonic dehyde) concentration and MPO (myeloperoxidase) activity were detected after 1 h of left lung ischemia, and lung tissue was evaluated by HE staining. The degree of pathological injury, and the ability of 3-MA to inhibit autophagy, and the analysis of autophagy in the lung ischemia-reperfusion The apoptotic cell ratio in the lung tissue was detected by the terminal dUTP nick end-labeling assay in the 3-MA treatment group and the control group, and the autophagy was analyzed to participate in the lung ischemia-reperfusion. Results: In the model of ischemia-reperfusion injury in the left lung of rats, the level of autophagy increased (P0.05) in the ischemia-reperfusion injury model, then the peak was reached at 2-6 h (P0.01), and the reperfusion group was restored to the near-sham operation group for 24 h. The electron microscopic examination was mainly in type 鈪，
本文编号：2491792
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