【摘要】：背景：肺缺血再灌注損傷是肺移植、袖式肺葉手術(shù)、心臟外科手術(shù)、心肺復(fù)蘇后綜合征等疾病中常見的引起不良后果的病理生理過(guò)程。以肺移植為例,有研究證實(shí),急慢性肺移植排斥反應(yīng)的發(fā)生均與肺移植術(shù)后早期肺缺血再灌注損傷的程度有關(guān)。已有的研究表明,肺缺血再灌注損傷主要表現(xiàn)為肺間質(zhì)水腫、炎癥細(xì)胞浸潤(rùn)、呼吸膜斷裂和氣體交換障礙。然而,肺缺血再灌注損傷發(fā)生發(fā)展的整體機(jī)制仍未被全面了解。 自噬是真核生物進(jìn)化高度保守的細(xì)胞自穩(wěn)程序。一方面,雙膜結(jié)構(gòu)的自噬囊泡能包繞隔絕失功能的細(xì)胞器、結(jié)構(gòu)破壞的蛋白質(zhì)和侵入細(xì)胞的病原體等。另一方面,自噬體與溶酶體結(jié)合消化囊內(nèi)物進(jìn)而為細(xì)胞代謝提供底物。生理狀態(tài)下細(xì)胞內(nèi)一定水平的自噬對(duì)維持細(xì)胞內(nèi)環(huán)境的穩(wěn)定起重要作用。當(dāng)被過(guò)度誘導(dǎo)時(shí),自噬也能導(dǎo)致細(xì)胞程序性死亡,與凋亡不同,這一過(guò)程被稱之為Ⅱ型程序性死亡。另外,由于自噬與凋亡的復(fù)雜聯(lián)系,一定程度的自噬甚至直接誘導(dǎo)細(xì)胞凋亡。因此,細(xì)胞自噬被認(rèn)為是細(xì)胞在壓力條件下的適應(yīng)機(jī)制。 國(guó)內(nèi)外越來(lái)越多的研究發(fā)現(xiàn)自噬在許多病理生理學(xué)狀態(tài)中扮演重要作用,例如腫瘤、感染、神經(jīng)退行性疾病等。國(guó)外有研究證實(shí)缺血再灌注過(guò)程能夠誘導(dǎo)自噬水平的快速上調(diào)。Chigure Suzuki等人的研究認(rèn)為自噬參與了腎臟的缺血再灌注損傷。然而,Man Jiang等在另一個(gè)腎臟缺血再灌注損傷模型中發(fā)現(xiàn)自噬在腎臟缺血再灌注損傷中是一種腎臟保護(hù)因素。在肝臟中,Kunihito Gotoh等發(fā)現(xiàn)藥物抑制自噬能夠改善肝移植術(shù)后的肝臟缺血再灌注損傷程度。因此,有學(xué)者推測(cè)自噬在缺血再灌注損傷中的作用可能是組織和模型依耐性的。當(dāng)前,自噬較少涉及呼吸系統(tǒng)疾病的研究,僅在吸煙、COPD、肺動(dòng)脈高壓等慢性呼吸系統(tǒng)疾病的肺組織中發(fā)現(xiàn)自噬水平升高,高水平的自噬與細(xì)胞的凋亡,組織纖維化與重構(gòu)密切相關(guān)。許多學(xué)者預(yù)測(cè)：自噬將成為肺部疾病基礎(chǔ)研究中一個(gè)新的前沿領(lǐng)域。那么,肺缺血再灌注時(shí)肺內(nèi)細(xì)胞自噬水平如何?自噬在肺缺血再灌注損傷中作用如何?本研究試圖在大鼠左肺原位熱缺血再灌注損傷模型中尋找答案。 目的：建立大鼠左肺原位熱缺血再灌注損傷模型,觀測(cè)大鼠肺缺血再灌注后肺組織內(nèi)細(xì)胞自噬水平的變化,探討肺缺血及再灌注對(duì)細(xì)胞自噬流的影響。藥物干預(yù)自噬,研究自噬在肺缺血再灌注損傷中的作用,并初步探索自噬在肺缺血再灌注損傷中發(fā)揮作用的可能機(jī)制。 方法：以SPF級(jí)SD大鼠作為實(shí)驗(yàn)動(dòng)物,建立左肺原位熱缺血再灌注損傷模型。運(yùn)用免疫印跡法檢測(cè)缺血1h和再灌注0、2、6、24h后肺組織內(nèi)自噬標(biāo)志蛋白LC3表達(dá)水平,免疫熒光染色法雙標(biāo)自噬體標(biāo)志蛋白LC3與溶酶體膜蛋白LAMP2,鑒定自噬體與溶酶體融合降解是否正常,電子顯微鏡觀測(cè)細(xì)胞內(nèi)自噬體,分析大鼠左肺缺血再灌注后肺組織內(nèi)細(xì)胞自噬水平變化情況。比較缺血3h、缺血1h再灌注2h、陽(yáng)性對(duì)照組、假手術(shù)組自噬水平,分析缺血和再灌注對(duì)自噬的誘導(dǎo)作用。自噬抑制劑3-甲基腺嘌呤(3-MA)和安慰劑分別預(yù)處理大鼠,檢測(cè)左肺缺血1h再灌注2h后肺干濕重比(Lung W/DRatio),MDA (malondialdehyde)濃度,MPO (myeloperoxidase)活性等肺損傷指標(biāo),HE染色評(píng)價(jià)肺組織病理學(xué)損傷程度,并驗(yàn)證3-MA抑制細(xì)胞自噬能力,分析自噬在肺缺血再灌注損傷中的作用。免疫印跡法檢測(cè)3-MA處理組和對(duì)照組肺組織中凋亡關(guān)鍵蛋白cleaved caspas-3表達(dá)水平,TUNEL (terminal dUTP nick end-labeling assay)法檢測(cè)肺組織中凋亡細(xì)胞比率,分析自噬參與肺缺血再灌注損傷的可能機(jī)制。 結(jié)果：在大鼠左肺熱缺血再灌注損傷模型中,自噬水平在缺血1h即有升高(P0.05),再灌注2-6h達(dá)到高峰(P0.01),再灌組24h恢復(fù)至接近假手術(shù)組水平(P0.05),電子顯微鏡檢查主要在Ⅰ型肺泡上皮細(xì)胞和血管內(nèi)皮細(xì)胞內(nèi)觀察到自噬體。缺血1h再灌注2h較缺血3h自噬水平高,與雷帕霉素處理的陽(yáng)性對(duì)照組自噬水平相當(dāng)。30mg/kg3-MA預(yù)處理與對(duì)照組比較降低肺組織損傷評(píng)分(P0.05),降低肺W/D,降低肺組織中MDA濃度和MPO活性。Western Blot結(jié)果示30mg/kg3-MA能有效抑制細(xì)胞自噬。同時(shí),與對(duì)照組比較,3-MA預(yù)處理降低肺組織中cleaved caspas-3蛋白水平,降低凋亡細(xì)胞比率。 結(jié)論：肺缺血和再灌注過(guò)程均顯著誘導(dǎo)肺組織細(xì)胞自噬水平上調(diào)。自噬藥物性抑制劑3-MA預(yù)處理能夠減輕肺缺血再灌注損傷程度,說(shuō)明自噬在肺缺血再灌注中有加重?fù)p傷的作用。其機(jī)制可能與高水平的細(xì)胞自噬誘導(dǎo)細(xì)胞凋亡,損傷肺呼吸膜有關(guān)。
[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，

本文編號(hào)：2491792