mtDNA及NETs在海水吸入性肺损伤中的作用机制研究

发布时间：2018-03-14 14:07

  本文选题：急性呼吸窘迫综合征　切入点：急性肺损伤　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:淹溺是一个严重的社会安全问题,每年都会造成大量的人员伤亡。作为淹溺的严重并发症,约有三分之一的受害者会并发急性肺损伤(acute lung injury,ALI)或是急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS),其也成为了淹溺导致死亡的重要原因之一。在所有种类的淹溺所致的肺损伤中又以海水淹溺导致的最为严重也最难以救治。然而关于海水淹溺性肺损伤缺乏详实的理论与基础研究,其发病机制及其与其它原因致肺损伤的异同也并不清楚,亟待进一步的深入研究。海水是一种低温、高渗、高钠、高钾、高钙、含多种细菌的碱性液体,其各种离子成分的浓度都明显异于淡水和人体体液,其渗透压约为人血浆渗透压的三倍(942vs.300m Osm/kg)。海水吸入后低温与高渗透压会联合作用在肺脏,对肺组织和细胞产生强烈的损伤性刺激。而且由于海水的渗透压高于血浆,肺泡周围间质及毛细血管中的液体会大量渗出到肺泡腔内,导致肺水肿和低氧血症的发生。线粒体损伤相关分子模式(mitochondria damage-associated molecular pattern,mt DAMP)是机体在受到强烈刺激或是创伤时机体所产生的来源于线粒体的一类物质。mt DAMP主要包括线粒体DNA和甲酰肽。有临床研究表明ICU中因脓毒血症和ARDS而死亡的患者血液中的游离mtDNA水平显著高于好转的患者,而且血液中mtDNA浓度高的病人死亡风险高于其他病人。mtDNA本身作为一种DAMP也可以起到诱发炎症反应的作用。mtDNA不同于细胞核DNA,它上面并没有许多甲基化修饰的Cp G基元(Cp G motifs),这一点与细菌DNA类似。机体的免疫系统可以识别出这种差异,从而激活免疫反应。mtDNA可以被免疫细胞、血管内皮细胞或是平滑肌细胞上的TLR9识别,进而促进前炎症因子的释放和MAPK通路的激活。在动物实验中,mtDNA静脉注射足以引起急性肺损伤的发生。其通过TLR9受体激活中性粒细胞,在急性肺损伤的发生和前期进展环节中起着重要的作用。中性粒细胞胞外杀菌网络(neutrophil extracellular traps,NETs)是中性粒细胞的一种杀菌机制,主要由DNA、髓过氧化物酶(myeloperoxidase,MPO)和组蛋白组成。NETs呈平滑的丝状结构,数根丝可以编制成束,继而这些束可以相互编制成网,在显微镜下观察类似于云状。NETs的存在便于在感染局部提供高浓度的抗菌物质,并可以增加它们的抗菌活性。NETs的产生也有着负面的影响。NETs参与了一些非感染性疾病的发生,如妊娠期高血压(子痫前期)以及系统性红斑狼疮。过量的中性粒细胞浸润以及NETs的形成也参与了急性肺损伤及急性呼吸窘迫综合征的病情的进展。在一项输血相关肺损伤的动物实验中,在肺的微循环中可以检测到NETs的生成。将生成的NETs加入到体外培养的肺上皮细胞中后,可以引起肺上皮细胞的凋亡。而且实验也显示NETs对肺内皮细胞同样具有毒性。c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)是MAPK通路下的一个分支,在细胞受到刺激时磷酸化并且激活,转移到线粒体或是细胞核,从而起到调控细胞功能的作用。其中JNK-线粒体通路参与了ROS的产生以及细胞凋亡的调控。实验表明JNK磷酸化激活后一部分转移到了细胞核,还有一部分通过与Sab的结合定位到了线粒体上。通过合成可以与Sab特异性结合的多肽Tat-SabKIM1可以通过一个简便的方法高效地阻断Sab与JNK相互结合和影响,继而抑制JNK向线粒体的定位,起到阻断JNK-线粒体通路,维护线粒体的正常功能。实验目的:1.研究海水吸入导致mtDNA生成的及其损伤作用。2.研究海水吸入及mtDNA产生导致NETs及其损伤作用。3.证实抑制JNK-线粒体通路激活可以通过减少ROS的产生继而影响mtDNA及NETs生成及作用,从而可以改善肺损伤。实验方法:第一部分:在本部分实验我们通过气道内滴注配方海水建立了海水吸入型肺损伤的大鼠模型,并进行了一系列的检测。首先我们通过留取肺组织测定了肺组织的湿干比,其代表了肺水肿的程度。我们还对肺组织进行了固定切片及HE染色,在显微镜下观察,评价了肺组织结构的损伤程度。接下来我们对留取的大鼠肺泡灌洗液进行了检测,测定了其中的蛋白含量和中性粒细胞数量,并通过q PCR的方法检测了各组大鼠肺泡灌洗液中mtDNA的含量。为进一步研究mtDNA对于肺组织及细胞的损伤作用,我们分离了大鼠心肌细胞的线粒体,并提取了其中的mtDNA用于对大鼠肺脏的刺激。我们还通过培养HUVEC细胞和A549细胞,在细胞层面做了海水刺激引起mtDNA释放的实验,并且检测了mtDNA对于细胞的损伤和单层细胞通透性的影响。我们还分离培养了健康志愿者全血中的中性粒细胞,并检测了mtDNA对于中性粒细胞黏附渗出及趋化功能的影响。除此之外,我们还通过蛋白质芯片的方法检测了中性粒细胞在受到mtDNA刺激时细胞因子的释放情况。第二部分:在这部分实验中我们主要检测了海水及mtDNA的刺激对于大鼠肺脏中NETs形成的影响。我们通过三种方法对NETs进行了检测,首先我们通过病理切片HE染色的方式观察了肺泡腔及肺组织中的细胞外游离的DNA,其次我们通过荧光染色的方法检测了肺泡灌洗液当中细胞外游离DNA的含量,除此之外我们通过ELISA的方法检测了Neutrophil elastase-DNA复合物的含量。我们还通过PMA诱导了并分离了NETs,并用产生的NETs刺激了体外培养的HUVEC及A549细胞,检测了NETs对于细胞的损伤及单层细胞通透性的影响。最后我们还应用了NAC抑制ROS的产生,检测了抗氧化剂对于NETs生成的影响。第三部分:在这部分实验中我们分离了肺组织中的线粒体蛋白和细胞质/细胞核蛋白,并通过Western blots的方法检测了p-JNK及JNK的表达情况,研究了海水刺激时JNK的转移情况。我们还检测了应用Tat-SabKIM1阻断线粒体—JNK通路后肺损伤情况及ROS的生成。实验结果:第一部分:1.在海水吸入型肺损伤的大鼠模型中可以在肺泡灌洗液中检测到mtDNA的存在。2.在海水刺激的HUVC细胞及A549细胞培养上清液中可以检测到mtDNA的存在。3.气管内滴注mtDNA可以诱导急性肺损伤的发生,而抑制mtDNA的产生可以起到改善急性肺损伤的作用。4.mtDNA可以引起HUVEC细胞及A549细胞的损伤及单层细胞通透性的改变。5.mtDNA可以激活中性粒细胞,促进中性粒细胞的黏附、渗出及迁移趋化能力,促进中性粒细胞的细胞因子释放。6.mtDNA的刺激可以诱导SD大鼠肺脏及相关细胞产生大量的ROS。第二部分:1.海水和mtDNA吸入可以引起SD大鼠肺脏产生无菌性NETs。2.海水刺激共培养的中性粒细胞及血管内皮细胞可以引起NETs的产生。3.产生的NETs可以引起HUVEC细胞及A549单层细胞通透性的改变。4.NETs的产生依赖于ROS,给予抗氧化剂后NETs的产生明显减少。第三部分:1.抑制JNK的线粒体转移可以通过减少ROS的产生起到改善肺损伤的作用。2.急性肺损伤时存在着JNK向线粒体转移的现象。3.应用Tat-SabKIM1多肽可以抑制JNK线粒体转移的现象。4.应用Tat-SabKIM1后可以抑制ROS的产生,起到改善肺损伤的作用。结论:本实验证实海水吸入可以刺激肺脏的组织细胞,扰乱细胞及线粒体的正常结构及功能,导致产生出大量的mtDNA并释放到肺泡腔及肺组织间隙中。释放的这些mtDNA可以损伤肺脏的细胞、破坏血气屏障,也可以凭借自身的免疫原性激活机体的免疫系统,引起中性粒细胞的黏附、渗出以及炎性因子的释放。mtDNA所引起的过度激活的免疫系统,尤其是激活中性粒细胞,进一步加重了肺损伤的程度。NETs是中性粒细胞所释放的一类胞外杀菌物质,在海水或是mtDNA的刺激下,大鼠的肺脏中会形成大量的NETs,这些NETs中存在着高浓度的炎症因子及细胞杀伤物质,可以对肺组织细胞造成损害,也可以起到加重肺损伤的作用。实验证实mtDNA和NETs的形成及产生损伤作用都是依赖于ROS的,在肺组织细胞处于氧化应激状态时,更易受到mtDNA及NETs的破坏。而JNK-线粒体通路的激活可以引起线粒体功能紊乱,造成ROS在细胞内的积聚。通过特异性多肽Tat-SabKIM1可以抑制海水刺激时肺脏细胞的JNK向线粒体转移,继而维持线粒体的稳定,阻断JNK-线粒体通路激活,起到抑制细胞自噬及凋亡、减轻肺损伤的作用。
[Abstract]:Background: drowning is a serious social security problem, will cause a lot of casualties every year. As a serious complication of drowning, about 1/3 of the victims of acute lung injury (acute lung, injury, ALI) or acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS), the drowning has become one of the leading causes of death in lung injury. All species caused by drowning in seawater drowning in the most serious and most difficult to treat. However on seawater drowning induced acute lung injury and the lack of theoretical basis of detailed research, the similarities and differences of lung injury caused by its pathogenesis and other the reason is not clear, need further study. The sea is a kind of low temperature, high permeability, high potassium, sodium, calcium, alkaline liquid containing a variety of bacteria, the concentration of various ions are significantly different from the fresh water And body fluids, the osmotic pressure is about three times of human plasma osmotic pressure (942vs.300m Osm/kg). After seawater inhalation of low temperature and high osmotic pressure joint role in lung injury, strong stimulation of lung tissue and cells. But due to the infiltration of sea water pressure is higher than the plasma, matter and liquid in the surrounding alveolar capillaries will exudation into the alveolar cavity, causing pulmonary edema and hypoxemia. Mitochondrial damage associated molecular patterns (mitochondria damage-associated molecular pattern, MT DAMP) is in the body by strong stimulation or is a kind of material.Mt DAMP in mitochondrial sources trauma body produced include mitochondrial DNA and formyl peptide. The clinical study showed that died of sepsis and ARDS ICU in the blood of patients with free mtDNA level was significantly higher than that of the patients improved, and the concentration of mtDNA in patients with high blood The risk of death was higher than that of other patients.MtDNA itself as a DAMP can also play a role in the inflammatory response induced by.MtDNA is different from the nuclear DNA, it did not have a lot of methylation Cp G motif (Cp G motifs), and this bacteria is similar to DNA. The immune system can recognize this difference. In order to activate the immune response of.MtDNA can be immune cells, vascular endothelial cells and smooth muscle cells on TLR9 recognition, and then promote the activation of MAPK pathway and release of proinflammatory cytokines. In animal experiments, mtDNA intravenous injection is sufficient to cause the occurrence of acute lung injury. The activation of TLR9 receptors in neutrophils, plays an important role in the occurrence of acute lung injury and early progress process. Neutrophil extracellular bactericidal network (neutrophil extracellular traps, NETs) is a kind of bactericidal mechanism of neutrophils, mainly by DNA, Myeloperoxidase (myeloperoxidase, MPO) and group.NETs protein composition of filamentous structure smooth, the number of threads can be woven into a bundle, then the beam can be compiled into a network, similar to the observation of cloud.NETs under a microscope for the presence of antimicrobial substances to provide convenient for local high levels of infection, and they can increase the antibacterial activity of.NETs have a negative impact on.NETs involved in some non infectious diseases, such as gestational hypertension (preeclampsia) and systemic lupus erythematosus. Progress of neutrophil infiltration and excess disease NETs formation is also involved in acute lung injury and acute respiratory distress syndrome in one. Animal experiment of transfusion associated lung injury, microcirculation in the lung was detected in the NETs generation. The generated NETs is added to the cultured lung epithelial cells, can cause lung epithelial The apoptosis of cells. And the experiments also showed that NETs has the same toxicity of.C-Jun N-terminal kinase in pulmonary endothelial cells (c-Jun N-terminal kinase, JNK) is a branch of the MAPK pathway, when cells are stimulated the phosphorylation and activation, is transferred to the nucleus or to mitochondria, regulation of cell function. JNK- the mitochondrial pathway is involved in the regulation of ROS generation and apoptosis. The experimental results showed that the phosphorylated JNK part of the transfer to the nucleus, and part combined with the localization of Sab to mitochondria. The polypeptide Tat-SabKIM1 synthesis can be combined with the specificity of Sab by a simple method and effectively blocking Sab JNK combined with each other and influence, and the inhibition of JNK to mitochondria localization, to blockade the JNK- mitochondrial pathway, maintain normal function of mitochondria. Objective: 1. to study water absorption The cause and damage effect of.2. on water generated by mtDNA inhalation and mtDNA lead to NETs and.3. confirmed the inhibition of JNK- damage can be reduced by activation of mitochondrial pathway of ROS production and effects of mtDNA and NETs formation and function, so it can improve the experimental lung injury. Method: the first part: in this part we through intratracheal note the formula established seawater seawater inhalation rat model of lung injury, and conducted a series of tests. We collected lung tissue were measured lung wet dry ratio, which represents the degree of pulmonary edema. We were fixed on the lung tissue sections and HE staining under the microscope. Next, evaluate the degree of lung injury structure. Next we take on the rat alveolar lavage fluid were detected, and the number of protein content of neutrophils was determined, and through Q PCR Methods to detect the content of bronchoalveolar lavage fluid of mtDNA rats in each group. For the further study of mtDNA for injury of lung tissue and cells, we isolated rat myocardial cell mitochondria, and extract the mtDNA for rat lung stimulation. We also through the cultivation of HUVEC cells and A549 cells in the cell. The level of mtDNA release caused by experimental stimulation of seawater, and tested the effects of mtDNA on cell injury and cell monolayer permeability. We also isolated neutrophils in whole blood of healthy volunteers, and to detect the effect of mtDNA on neutrophil adhesion to exudation and chemotactic function. In addition, we detected the release of neutrophil cells under stimulation by mtDNA factor through the method of protein chip. The second part: in this part we mainly detected in seawater and mtDNA The stimulation effect on NETs formation in rat lungs. We through the three methods for NETs were detected by HE staining, we observed the lung tissue and alveolar cavity in the cell free DNA method, then we detected by fluorescent staining in bronchoalveolar lavage and content of extracellular free lotion DNA, besides we detected by ELISA in Neutrophil elastase-DNA complexes. We also induced by PMA and separation of NETs, and used the resulting NETs stimulation of the cultured HUVEC and A549 cells to examine the effects of NETs on cell injury and cell monolayer permeability. Finally we also the application of NAC to inhibit the production of ROS, examined the antioxidant effects on NETs generation. The third part: in this part we isolated lung tissue in mitochondria and cytoplasm / The nuclear protein and detect the expression of p-JNK and JNK by Western blots, on the JNK of the seawater transfer stimulation. We also examined the application of Tat-SabKIM1 block and ROS lung injury - mitochondrial JNK pathway. Results: the first part: 1. in the seawater inhalation rat model of lung injury in the presence of mtDNA in bronchoalveolar lavage and detection of.2. in seawater stimulated HUVC cells and A549 cell culture supernatant was detected in the presence of.3. intratracheal instillation of mtDNA mtDNA can induce the occurrence of acute lung injury to the solution, the role of.4.mtDNA and inhibit the production of mtDNA can improve the acute lung injury can cause injury HUVEC cells and A549 cells and the cell monolayer permeability.5.mtDNA can activate neutrophils, promote the adhesion of neutrophils, exudation and migration ability, promote Cytokine release of neutrophil.6.mtDNA stimulation can induce SD rat lung cells to produce ROS. second and a large part of the inhalation can cause SD rats lung aseptic seawater NETs.2. stimulation of co cultured neutrophils and endothelial cells of 1. seawater and mtDNA can cause the generation of NETs.3. NETs cause HUVEC cell and A549 cell monolayer permeability of.4.NETs depends on ROS, give the production of NETs decreased obviously after antioxidants. The third part: 1. JNK inhibiting mitochondrial transfer by reducing ROS production to improve the role of.2. in acute lung injury injury when there is a phenomenon of.3. application of Tat-SabKIM1 JNK to the mitochondrial polypeptide the transfer of JNK can inhibit the mitochondrial transfer phenomena of.4. Tat-SabKIM1 could inhibit the production of ROS, to improve lung injury. Conclusion: the The experiment confirmed that seawater inhalation can stimulate lung tissue cells, the normal structure and function of cells and disrupt the mitochondria, resulting in a large number of mtDNA and released into the cavity of lung tissue and alveolar space. The release of these mtDNA can damage the lung cell damage, blood gas barrier, but also can rely on the immune system of their immunogenicity activate body the adhesion of neutrophils, caused by excessive activation of the immune system, the exudation and release of inflammatory factors induced by.MtDNA, especially the activation of neutrophils, further aggravate lung injury degree of.NETs is a kind of extracellular release of neutrophils and bactericidal substances in seawater or mtDNA stimulation rats in the lung will form a large number of NETs, the NETs has high concentrations of inflammatory cytokines and cell killing substances, can cause damage to the lung tissue, can also play worse Lung injury. The experiment demonstrated that mtDNA and NETs formed and negative effects are dependent on ROS, in lung tissue cells under oxidative stress, mtDNA and NETs are more susceptible to damage. And the activation of the JNK- mitochondrial pathway can cause mitochondrial dysfunction, resulting in ROS accumulation in the cells of the lung. The JNK cell transfer to seawater when stimulated by inhibition of mitochondrial specific peptide Tat-SabKIM1, and then maintain mitochondrial stability, blocking JNK- mitochondrial pathway activation, inhibit autophagy and apoptosis, reduce the lung injury.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R649.3;R563.8
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本文编号：1611539