本文選題：急性呼吸窘迫綜合征　切入點：急性肺損傷　出處：《第四軍醫(yī)大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景:淹溺是一個嚴重的社會安全問題,每年都會造成大量的人員傷亡。作為淹溺的嚴重并發(fā)癥,約有三分之一的受害者會并發(fā)急性肺損傷(acute lung injury,ALI)或是急性呼吸窘迫綜合征(acute respiratory distress syndrome,ARDS),其也成為了淹溺導致死亡的重要原因之一。在所有種類的淹溺所致的肺損傷中又以海水淹溺導致的最為嚴重也最難以救治。然而關于海水淹溺性肺損傷缺乏詳實的理論與基礎研究,其發(fā)病機制及其與其它原因致肺損傷的異同也并不清楚,亟待進一步的深入研究。海水是一種低溫、高滲、高鈉、高鉀、高鈣、含多種細菌的堿性液體,其各種離子成分的濃度都明顯異于淡水和人體體液,其滲透壓約為人血漿滲透壓的三倍(942vs.300m Osm/kg)。海水吸入后低溫與高滲透壓會聯(lián)合作用在肺臟,對肺組織和細胞產生強烈的損傷性刺激。而且由于海水的滲透壓高于血漿,肺泡周圍間質及毛細血管中的液體會大量滲出到肺泡腔內,導致肺水腫和低氧血癥的發(fā)生。線粒體損傷相關分子模式(mitochondria damage-associated molecular pattern,mt DAMP)是機體在受到強烈刺激或是創(chuàng)傷時機體所產生的來源于線粒體的一類物質。mt DAMP主要包括線粒體DNA和甲酰肽。有臨床研究表明ICU中因膿毒血癥和ARDS而死亡的患者血液中的游離mtDNA水平顯著高于好轉的患者,而且血液中mtDNA濃度高的病人死亡風險高于其他病人。mtDNA本身作為一種DAMP也可以起到誘發(fā)炎癥反應的作用。mtDNA不同于細胞核DNA,它上面并沒有許多甲基化修飾的Cp G基元(Cp G motifs),這一點與細菌DNA類似。機體的免疫系統(tǒng)可以識別出這種差異,從而激活免疫反應。mtDNA可以被免疫細胞、血管內皮細胞或是平滑肌細胞上的TLR9識別,進而促進前炎癥因子的釋放和MAPK通路的激活。在動物實驗中,mtDNA靜脈注射足以引起急性肺損傷的發(fā)生。其通過TLR9受體激活中性粒細胞,在急性肺損傷的發(fā)生和前期進展環(huán)節(jié)中起著重要的作用。中性粒細胞胞外殺菌網絡(neutrophil extracellular traps,NETs)是中性粒細胞的一種殺菌機制,主要由DNA、髓過氧化物酶(myeloperoxidase,MPO)和組蛋白組成。NETs呈平滑的絲狀結構,數(shù)根絲可以編制成束,繼而這些束可以相互編制成網,在顯微鏡下觀察類似于云狀。NETs的存在便于在感染局部提供高濃度的抗菌物質,并可以增加它們的抗菌活性。NETs的產生也有著負面的影響。NETs參與了一些非感染性疾病的發(fā)生,如妊娠期高血壓(子癇前期)以及系統(tǒng)性紅斑狼瘡。過量的中性粒細胞浸潤以及NETs的形成也參與了急性肺損傷及急性呼吸窘迫綜合征的病情的進展。在一項輸血相關肺損傷的動物實驗中,在肺的微循環(huán)中可以檢測到NETs的生成。將生成的NETs加入到體外培養(yǎng)的肺上皮細胞中后,可以引起肺上皮細胞的凋亡。而且實驗也顯示NETs對肺內皮細胞同樣具有毒性。c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)是MAPK通路下的一個分支,在細胞受到刺激時磷酸化并且激活,轉移到線粒體或是細胞核,從而起到調控細胞功能的作用。其中JNK-線粒體通路參與了ROS的產生以及細胞凋亡的調控。實驗表明JNK磷酸化激活后一部分轉移到了細胞核,還有一部分通過與Sab的結合定位到了線粒體上。通過合成可以與Sab特異性結合的多肽Tat-SabKIM1可以通過一個簡便的方法高效地阻斷Sab與JNK相互結合和影響,繼而抑制JNK向線粒體的定位,起到阻斷JNK-線粒體通路,維護線粒體的正常功能。實驗目的:1.研究海水吸入導致mtDNA生成的及其損傷作用。2.研究海水吸入及mtDNA產生導致NETs及其損傷作用。3.證實抑制JNK-線粒體通路激活可以通過減少ROS的產生繼而影響mtDNA及NETs生成及作用,從而可以改善肺損傷。實驗方法:第一部分:在本部分實驗我們通過氣道內滴注配方海水建立了海水吸入型肺損傷的大鼠模型,并進行了一系列的檢測。首先我們通過留取肺組織測定了肺組織的濕干比,其代表了肺水腫的程度。我們還對肺組織進行了固定切片及HE染色,在顯微鏡下觀察,評價了肺組織結構的損傷程度。接下來我們對留取的大鼠肺泡灌洗液進行了檢測,測定了其中的蛋白含量和中性粒細胞數(shù)量,并通過q PCR的方法檢測了各組大鼠肺泡灌洗液中mtDNA的含量。為進一步研究mtDNA對于肺組織及細胞的損傷作用,我們分離了大鼠心肌細胞的線粒體,并提取了其中的mtDNA用于對大鼠肺臟的刺激。我們還通過培養(yǎng)HUVEC細胞和A549細胞,在細胞層面做了海水刺激引起mtDNA釋放的實驗,并且檢測了mtDNA對于細胞的損傷和單層細胞通透性的影響。我們還分離培養(yǎng)了健康志愿者全血中的中性粒細胞,并檢測了mtDNA對于中性粒細胞黏附滲出及趨化功能的影響。除此之外,我們還通過蛋白質芯片的方法檢測了中性粒細胞在受到mtDNA刺激時細胞因子的釋放情況。第二部分:在這部分實驗中我們主要檢測了海水及mtDNA的刺激對于大鼠肺臟中NETs形成的影響。我們通過三種方法對NETs進行了檢測,首先我們通過病理切片HE染色的方式觀察了肺泡腔及肺組織中的細胞外游離的DNA,其次我們通過熒光染色的方法檢測了肺泡灌洗液當中細胞外游離DNA的含量,除此之外我們通過ELISA的方法檢測了Neutrophil elastase-DNA復合物的含量。我們還通過PMA誘導了并分離了NETs,并用產生的NETs刺激了體外培養(yǎng)的HUVEC及A549細胞,檢測了NETs對于細胞的損傷及單層細胞通透性的影響。最后我們還應用了NAC抑制ROS的產生,檢測了抗氧化劑對于NETs生成的影響。第三部分:在這部分實驗中我們分離了肺組織中的線粒體蛋白和細胞質/細胞核蛋白,并通過Western blots的方法檢測了p-JNK及JNK的表達情況,研究了海水刺激時JNK的轉移情況。我們還檢測了應用Tat-SabKIM1阻斷線粒體—JNK通路后肺損傷情況及ROS的生成。實驗結果:第一部分:1.在海水吸入型肺損傷的大鼠模型中可以在肺泡灌洗液中檢測到mtDNA的存在。2.在海水刺激的HUVC細胞及A549細胞培養(yǎng)上清液中可以檢測到mtDNA的存在。3.氣管內滴注mtDNA可以誘導急性肺損傷的發(fā)生,而抑制mtDNA的產生可以起到改善急性肺損傷的作用。4.mtDNA可以引起HUVEC細胞及A549細胞的損傷及單層細胞通透性的改變。5.mtDNA可以激活中性粒細胞,促進中性粒細胞的黏附、滲出及遷移趨化能力,促進中性粒細胞的細胞因子釋放。6.mtDNA的刺激可以誘導SD大鼠肺臟及相關細胞產生大量的ROS。第二部分:1.海水和mtDNA吸入可以引起SD大鼠肺臟產生無菌性NETs。2.海水刺激共培養(yǎng)的中性粒細胞及血管內皮細胞可以引起NETs的產生。3.產生的NETs可以引起HUVEC細胞及A549單層細胞通透性的改變。4.NETs的產生依賴于ROS,給予抗氧化劑后NETs的產生明顯減少。第三部分:1.抑制JNK的線粒體轉移可以通過減少ROS的產生起到改善肺損傷的作用。2.急性肺損傷時存在著JNK向線粒體轉移的現(xiàn)象。3.應用Tat-SabKIM1多肽可以抑制JNK線粒體轉移的現(xiàn)象。4.應用Tat-SabKIM1后可以抑制ROS的產生,起到改善肺損傷的作用。結論:本實驗證實海水吸入可以刺激肺臟的組織細胞,擾亂細胞及線粒體的正常結構及功能,導致產生出大量的mtDNA并釋放到肺泡腔及肺組織間隙中。釋放的這些mtDNA可以損傷肺臟的細胞、破壞血氣屏障,也可以憑借自身的免疫原性激活機體的免疫系統(tǒng),引起中性粒細胞的黏附、滲出以及炎性因子的釋放。mtDNA所引起的過度激活的免疫系統(tǒng),尤其是激活中性粒細胞,進一步加重了肺損傷的程度。NETs是中性粒細胞所釋放的一類胞外殺菌物質,在海水或是mtDNA的刺激下,大鼠的肺臟中會形成大量的NETs,這些NETs中存在著高濃度的炎癥因子及細胞殺傷物質,可以對肺組織細胞造成損害,也可以起到加重肺損傷的作用。實驗證實mtDNA和NETs的形成及產生損傷作用都是依賴于ROS的,在肺組織細胞處于氧化應激狀態(tài)時,更易受到mtDNA及NETs的破壞。而JNK-線粒體通路的激活可以引起線粒體功能紊亂,造成ROS在細胞內的積聚。通過特異性多肽Tat-SabKIM1可以抑制海水刺激時肺臟細胞的JNK向線粒體轉移,繼而維持線粒體的穩(wěn)定,阻斷JNK-線粒體通路激活,起到抑制細胞自噬及凋亡、減輕肺損傷的作用。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R649.3;R563.8
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本文編號：1611539