本文選題：機械通氣 + 肺損傷��； 參考：《南方醫(yī)科大學》2014年碩士論文

【摘要】：1研究背景和目的 機械通氣(MV)已成為救治呼吸衰竭的重要手段之一,但其本身也可誘發(fā)或加重原有的肺損傷,導致機械通氣肺損傷(VILI)。VILI的組織病理學改變和其他原因引起的肺損傷相似,均伴有肺泡上皮和血管內(nèi)皮的廣泛性破壞、肺泡毛細血管膜通透性增加、肺水腫、出血、透明膜形成和炎性細胞浸潤等病理學改變。自1974年,webb等人最早提出機械通氣能導致肺損傷這一概念至今[2], VILI日益受到人們的關注,近年來對VILI的發(fā)病機制進行了大量的研究。目前認為VILI主要是由于高氣道峰壓和大潮氣量通氣導致吸氣末肺組織過度擴張,以及不張的終末小氣道或肺泡隨機械通氣周期性開放和關閉所引起的機械性肺損傷,此外,在機械性損傷的基礎上,肺內(nèi)炎癥細胞聚集、活化和釋放炎性介質(zhì)可進一步促進VILI的發(fā)生和發(fā)展。根據(jù)VILI的損傷類型,一般將VILI大致分為以下幾種類型,即氣壓傷、容量傷、不張傷和生物傷。其中前兩者屬于機械性損傷,一般早期首先出現(xiàn)機械性損傷,隨后以炎癥細胞、細胞因子介導的生物傷為主,兩者相互聯(lián)系,生物傷作用重要,機制復雜,成為VILI的研究熱點。大量研究表明,機械通氣產(chǎn)生的異常增高的牽張、剪切力等機械刺激作用于肺細胞,導致肺細胞內(nèi)眾多信號轉(zhuǎn)導信號激活如MAPK通路、NF-κB系統(tǒng)以及細胞膜表面的牽拉敏感性離子通道,使各種炎性細胞如TNF-α IL-1β、IL-8等表達增多引起白細胞向肺組織浸潤,這些炎癥細胞和細胞因子相互作用,構成一個龐大且復雜的網(wǎng)絡體系,不但能直接導致肺組織損傷,還可通過血液循環(huán)釋放到全身各個組織器官,引起多器官功能障礙(MODS)甚至危及生命。 雖然在過去幾十年內(nèi)對VILI的發(fā)病機制有了大量的研究也取得了顯著的成果,但是其確切機制目前并不是很清楚。最近有人提出機械通氣肺損傷可能存在晝夜節(jié)律性,這為VILI的機制研究開辟了一條新的道路。晝夜節(jié)律是所有生物體對可預測的環(huán)境改變的一種綜合性的適應,它是可以持續(xù)運行并且以大約24h為周期的生物節(jié)律,是生物界最普遍的一種生物節(jié)律,與許多生理和行為過程有關,如睡眠-覺醒周期、激素水平、體溫、血壓、呼吸等。晝夜節(jié)律的中樞在下丘腦視交叉上核,也存在于機體的各個組織器官,肺組織也不例外。研究顯示,人體正常肺功能具有明顯的晝夜節(jié)律性,潮氣量、分鐘通氣量、平均吸氣速率以及呼吸效率等均存在晝夜節(jié)律性變化,時鐘基因在正常肺組織中呈規(guī)律表達以調(diào)節(jié)正常肺功能。更有研究表明,一些呼吸系統(tǒng)疾病如呼吸道感染、哮喘、非小細胞肺癌等同樣也具有一定的晝夜節(jié)律性。那么,作為呼吸系統(tǒng)相關疾病的VILI是否也具有晝夜節(jié)律性呢?目前國內(nèi)外鮮有報道。因此,本研究主要從晝夜節(jié)律這個新視角來探討VILI的發(fā)病機制,以期為VILI的防治提供一種新思路和藥物作用新靶點。 2材料和方法 2.1第一章：機械通氣肺損傷大鼠肺組織中核內(nèi)受體Rev-erba表達改變 清潔級同批SD大鼠,體重180-220g,24只,由中山大學實驗動物中心提供。隨機分為3組(n=8)：F組(自由呼吸組)、LV組(小潮氣量組)、HV組(大潮氣量組),在光照-黑暗(Light-dark, LD)交替條件下飼養(yǎng),動物房溫維持在20±2℃。采用LD光制(12h：12h)即12小時光照(光照期08：00-20：00)與12小時黑暗(黑暗期20:00-08：00)交替,適應性飼養(yǎng)2周后開始實驗。所有大鼠均用10%的水合氯醛0.35g/100g腹腔注射麻醉,仰臥位固定,行右頸總動脈、尾靜脈置管術,右頸總動脈置管。待大鼠對口咽部刺激無反應后經(jīng)口直視下插入14號靜脈套管針作為氣管導管,血壓穩(wěn)定后,從尾靜脈緩慢推入維庫溴胺(0.2mg/ml,生理鹽水稀釋)0.6mg/kg,自主呼吸消失后,連接小動物呼吸機行機械通氣。其中F組插管后不給肌松藥不行機械通氣讓大鼠自主呼吸,LV組給予10ml/kg小潮氣量行機械通氣,HV組給予40ml/kg大潮氣量行機械通氣,通氣時間為2h,機械通氣過程中維持泵入維庫溴胺(0.2mg/ml)20ml/kg/h。通氣完畢后,待自主呼吸恢復平穩(wěn)即停止通氣,拔除氣管導管以及動靜脈套管針,動脈結(jié)扎止血,靜脈壓迫止血,肌肉和皮膚對應縫合,置大鼠于動物房中單籠飼養(yǎng)。24小時后取大鼠肺組織檢測各組肺濕干重比值；HE染色觀察各組肺組織病理改變；RT-PCR和Western blot方法檢測Bmal1、Clock、Per2、 Rev-erba等時鐘基因mRNA和蛋白表達水平。 2.2第二章：特異激動劑SR9009對大潮氣量機械通氣肺損傷大鼠的治療效果 清潔級同批SD大鼠,體重180-220g,16只,由中山大學實驗動物中心提供。隨機分為2組(n=8):control組、SR9009組,在光照-黑暗(Light-dark, LD)交替條件下飼養(yǎng),動物房溫維持在20±2℃。采用LD光制(12h：12h)即12小時光照(光照期08：00-20：00)與12小時黑暗(黑暗期20：00-08：00)交替,適應性飼養(yǎng)2周后開始實驗。其中SR9009組在麻醉前腹腔注射50mg/kg SR9009,control組注射等量稀釋液DMSO,30min后兩組大鼠均用10%的水合氯醛0.35g/100g腹腔注射麻醉,仰臥位固定,行右頸總動脈、尾靜脈置管術,右頸總動脈置管。待大鼠對口咽部刺激無反應后經(jīng)口直視下插入14號靜脈套管針作為氣管導管,血壓穩(wěn)定后,從尾靜脈緩慢推入維庫溴胺(0.2mg/ml,生理鹽水稀釋)0.6mg/kg,自主呼吸消失后,連接小動物呼吸機行機械通氣。兩組均采用大潮氣量機械通氣,呼吸機參數(shù)設置為：VT=40ml/kg,f=40次／分,I：E=1：1.5,通氣時間為2h,機械通氣過程中維持泵入維庫溴胺(0.2mg/ml)20ml/kg/h。通氣完畢后,待自主呼吸恢復平穩(wěn)即停止通氣,拔除氣管導管以及動靜脈套管針,動脈結(jié)扎止血,靜脈壓迫止血,肌肉和皮膚對應縫合,置大鼠于動物房中單籠飼養(yǎng)。24小時后取大鼠肺組織檢測各組肺濕干重比值；HE染色觀察兩組肺組織病理改變；Elisa實驗對比兩組肺組織TNF—α含量變化。 3結(jié)果 3.1第一章結(jié)果 3.1.1大鼠肺組織病理改變 肺組織病理結(jié)果顯示,自由呼吸組大鼠肺臟外觀正常,光鏡下可見肺組織結(jié)構完整、肺泡腔清晰、肺泡間質(zhì)無水腫,無炎性浸潤改變；小潮氣量組大鼠肺臟外觀略顯腫脹,但表面色澤基本正常,光鏡下可見輕度肺間質(zhì)水腫和少量巨噬細胞、淋巴和單核細胞等炎癥細胞浸潤；大潮氣量組大鼠肺臟外觀明顯腫脹,表面可見點狀出血,光鏡下可見彌漫性肺間質(zhì)水腫,肺泡腔、血管旁和支氣管周圍有大量炎性細胞浸潤,肺泡間隔明顯增厚。 3.1.2肺濕干重比值 肺濕干重比值(W/D值)是評價肺水腫的指標,與對照組比較,小潮氣量組大鼠肺組織W/D值略微增加；與對照組、小潮氣量兩組相比,大潮氣量組大鼠機械通氣2h后,W/D值明顯增加,(P0.05),說明大潮氣量組機械通氣后造成嚴重肺水增多,肺水腫。 3.1.3Bmal1、Clock、Per2、Rev-erba等時鐘基因mRNA表達水平 與自由呼吸組和小潮氣量組相比,大潮氣量組大鼠肺組織Bmal1、Clock基因mRNA略微升高但無顯著性差異；Per2基因mRNA略微下降但無顯著性差異。與自由呼吸組相比,小潮氣量組Rev-erbα基因mRNA呈顯著性下降,且有統(tǒng)計學意義(P0.05),與自由呼吸組和小潮氣量組相比,大潮氣量組大鼠肺組織Rev-erbα基因mRNA呈顯著性下降,且有統(tǒng)計學意義(P0.05)。 3.1.4Rev-erbα蛋白表達水平 與自由呼吸組相比,小潮氣量組大鼠肺組織Rev-erbα蛋白產(chǎn)物表達輕微下調(diào),大潮氣量組表達顯著下調(diào),且有統(tǒng)計學意義(P0.05)。 3.1.5炎癥因子TNF-α濃度變化 與自由呼吸組相比,小潮氣量組大鼠肺組織TNF-α表達顯著上調(diào),P0.05；大潮氣量組表達顯著上調(diào),P0.001。 3.2第二章結(jié)果 3.2.1肺組織病理學檢測結(jié)果 Control組大鼠肺組織與大潮氣量組機械通氣大鼠肺組織改變相似,大鼠肺臟外觀明顯腫脹,表面可見點狀出血,光鏡下可見肺泡腔、血管旁、支氣管周圍有大量炎性細胞浸潤,肺泡壁增厚,彌漫性肺間質(zhì)出血和水腫；SR9009組大鼠肺臟外觀略顯腫脹,但表面色澤基本正常,光鏡下可見少量炎癥細胞浸潤和輕微肺水腫,與小潮氣量組機械通氣大鼠肺組織改變相似。該結(jié)果說明SR9009能明顯減輕大潮氣量機械通氣大鼠肺損傷程度并有效改善肺組織炎癥反應。 3.2.2肺組織TNF-α含量 與Control組比較,大鼠肺組織TNF-α表達水平明顯降低,且有統(tǒng)計學意義(P0.05)。該結(jié)果說明SR9009能明顯降低大潮氣量機械通氣大鼠肺組織腫瘤壞死因子α水平,減輕肺組織炎癥反應。 4結(jié)論 (1)大潮氣量機械通氣時由于機械牽拉導致肺泡上皮細胞損傷或壞死,并誘導肺內(nèi)炎性細胞因子的釋放,從而導致機械通氣肺損傷,本研究成功模擬了大潮氣量所致機械通氣肺損傷動物模型； (2)大鼠機械通氣所致肺損傷可能發(fā)生肺組織晝夜節(jié)律紊亂；Rev-erbα在大鼠機械通氣肺損傷中發(fā)揮重要作用； (3) Rev-erbα蛋白特異激動劑SR9009對機械通氣肺損傷具有良好的治療效果,能顯著減輕大潮氣量機械通氣大鼠肺損傷程度,降低大潮氣量所致TNF-a水平,減輕肺組織炎癥反應。
[Abstract]:1 background and purpose of research
Mechanical ventilation (MV) has become one of the most important means for the treatment of respiratory failure, but it can also induce or aggravate the original lung injury, resulting in mechanical ventilation injury (VILI).VILI histopathological changes similar to other causes of lung injury, accompanied by the extensive destruction of alveolar epithelium and vascular endothelium, alveolar capillary membrane Increase in permeability, pulmonary edema, bleeding, hyaline membrane formation and inflammatory cell infiltration. Since 1974, Webb et al. First proposed that the concept of mechanical ventilation can lead to lung injury is [2], and VILI has been paid more attention to. In recent years, a lot of studies have been made on the pathogenesis of VILI. At present, it is believed that VILI is mainly due to high airway. Peak pressure and tidal volume ventilation lead to excessive expansion of the terminal lung tissue, and the mechanical lung injury caused by the opening and closing of the inflexible terminal small airway or alveoli with the periodic opening and closing of the mechanical ventilation. In addition, on the basis of mechanical damage, the accumulation of inflammatory cells in the lungs, activation and release of inflammatory mediators can further promote the occurrence of VILI. Development. According to the damage type of VILI, VILI is generally divided into the following types, namely, air pressure injury, volume injury, inelectate injury and biological injury. The first two belong to mechanical damage. Generally, the first two are mechanical damage. In general, early mechanical damage is first appeared, followed by biological injury mediated by inflammatory cells and cytokines, the two are interrelated and biological injury is important, A large number of studies have shown that the abnormal increase of mechanical ventilation induced by mechanical ventilation, such as distraction, shear force and other mechanical stimulation on lung cells, leads to the activation of many signal transduction signals in the lung cells, such as MAPK pathway, NF- kappa B system and the sensitive ion channel on the surface of the cell membrane to make various inflammatory cells such as TNF-, such as TNF-. The increase of the expression of alpha IL-1 beta and IL-8 causes leukocytes to infiltrate to the lung tissue. These inflammatory cells and cytokines interact and form a huge and complex network system. It can not only directly cause lung tissue damage, but also can be released to all tissues and organs through blood circulation, causing multiple organ dysfunction (MODS) and even life-threatening.
Although considerable research has been made on the pathogenesis of VILI in the past few decades, the exact mechanism is not very clear. Recently, it is suggested that circadian rhythms may exist in mechanical ventilated lung injury, which opens up a new way for the study of the mechanism of VILI. A comprehensive adaptation of predictable environmental changes, a biological rhythm that can continue to run and cycle around 24h, is one of the most common biological rhythms in the biological community, related to many physiological and behavioral processes, such as the sleep wake cycle, hormone levels, body temperature, blood pressure, respiration, and so on. The center of the circadian rhythm in the hypothalamus is seen in the hypothalamus. The study shows that the normal lung function of the human body has an obvious circadian rhythm, the tidal volume, the minute ventilation, the average breathing rate and the respiratory efficiency are all circadian rhythms, and the clock gene is regularly expressed in normal lung to regulate the normal lung. More studies have shown that some respiratory diseases, such as respiratory infection, asthma, and non small cell lung cancer, also have a certain circadian rhythm. Then, is VILI as a respiratory related disease also circadian rhythms? There are few reports at home and abroad. Therefore, this new perspective is mainly from the circadian rhythm. Objective to explore the pathogenesis of VILI in order to provide a new idea and a new target for the prevention and treatment of VILI.
2 materials and methods
2.1 Chapter 1: changes of receptor Rev-erba expression in lung tissue of rats with lung injury induced by mechanical ventilation
The same batch of SD rats, weight 180-220g and 24, were provided by the experimental animal center of Zhongshan University. They were randomly divided into 3 groups (n=8):F group (free breathing group), LV group (small tidal volume group), HV group (tidal volume group), under the alternate conditions of light dark (Light-dark, LD), the room temperature of the animal was maintained at 20 + 2 degrees. LD light (12h:12h) was used for 12 hours. Light (light period 08:00-20:00) and 12 hours of dark (dark period 20:00-08:00) alternate, adaptive feeding 2 weeks after the experiment. All rats were injected with 10% of chloral chloral 0.35g/100g intraperitoneal anesthesia, supine position fixed, the right cervical artery, the tail vein catheterization, the right cervical artery catheterization. The 14 vein cannula was inserted into the tracheal tube as the tracheal tube. After the blood pressure was stable, 0.6mg/kg was slowly pushed into the vecuronium bromide from the caudal vein (0.2mg/ml, diluted with physiological saline). After the spontaneous breathing disappeared, the small animal ventilator was connected with mechanical ventilation. In group F, no muscle relaxants were not given mechanical ventilation to breathe spontaneously in rats, and group LV was given 10ml/ The volume of kg was ventilated by mechanical ventilation, and group HV was given mechanical ventilation with the volume of 40ml/kg, and the duration of ventilation was 2H. After mechanical ventilation, it was maintained that the pump entered the VVB (0.2mg/ml) 20ml/kg/h. ventilation after the ventilation was completed, and the spontaneous breathing was stopped to stop ventilation, the tracheal catheter was removed and the venous cannula was extracted, the arterial ligature was stopped, and the venous oppression stopped bleeding. The muscle and skin were sutured, and rats were reared in the animal house for.24 hours. The lung wet dry weight ratio of each group was detected by the rat lung tissue. The pathological changes of lung tissues were observed by HE staining. RT-PCR and Western blot methods were used to detect the expression of mRNA and protein of Bmal1, Clock, Per2, Rev-erba.
2.2 second chapter: therapeutic effect of specific agonist SR9009 on rats with lung injury induced by tidal volume ventilation
The same batch of SD rats, weight 180-220g and 16, were provided by the experimental animal center of Zhongshan University. They were randomly divided into 2 groups (n=8): control group, SR9009 group, under the alternate conditions of light dark (Light-dark, LD), the room temperature of the animal was maintained at 20 + 2 degrees C. LD light (12h:12h) was 12 small hours (light period 08:00-20:00) and 12 hours dark ( 20:00-08:00 in the dark period was alternated, and the experiment was started after 2 weeks of adaptation. In group SR9009, 50mg/kg SR9009 was intraperitoneally injected before anesthesia, and DMSO in group control was injected with equal amount of diluent. After 30min, the two groups of rats were anesthetized with 10% hydrous chloral 0.35g/100g intraperitoneally, and the supine position was fixed. Right cervical artery, caudal vein catheterization and right common carotid artery were performed. After the rats had no reaction to the oropharynx stimulation, the rats were inserted into the 14 vein cannula as the tracheal tube, and after the blood pressure was stable, 0.6mg/kg was slowly pushed into the vecuronium bromide from the tail vein (0.2mg/ml, the saline diluted). After the spontaneous breathing disappeared, the small animal breathing machine was ventilated by mechanical ventilation. The two groups were all ventilated by large tidal volume. The parameters of the ventilator were as follows: VT=40ml/kg, f=40 times / sub, I:E=1:1.5, the duration of ventilation was 2h, and during the mechanical ventilation, after the maintenance of the pump into the VVB (0.2mg/ml) 20ml/kg/h., the ventilation was stopped, the tracheal catheter and the venous cannula were removed, the artery ligation, the venous compression hemostasis, the muscle and the muscle were used. The skin was sutured, and the rat lung was reared in the animal house for.24 hours. The lung wet dry weight ratio was measured in the lung tissue of the rats. The pathological changes of the two groups of lung tissues were observed by HE staining, and the changes of the TNF - alpha content in the lung tissue were compared with the Elisa test.
3 Results
3.1 chapter one results
Pathological changes of lung tissue in 3.1.1 rats
The lung tissue pathological results showed that the lungs of the free breathing rats were normal, the lung tissue was intact, the alveolus was clear, the alveolar interstitial was no edema and no inflammatory infiltration. The lung appearance of the rats in the small tidal volume group was slightly swollen, but the surface color was basically normal. Light interstitial edema and a small number of macrophages were seen under light microscope. Inflammatory cells such as lymphatic and mononuclear cells were infiltrated, and the lung appearance of rats in the large tidal volume group was obviously swollen, superficial bleeding was visible, diffuse interstitial edema, alveolar cavity, large number of inflammatory cells around the blood vessel and around the bronchi, and the alveolar septum thickened obviously.
3.1.2 ratio of wet dry weight of lung
The ratio of lung wet dry weight (W/D) was the index for evaluating pulmonary edema. Compared with the control group, the W/D value of lung tissue of rats in the small tidal volume group increased slightly. Compared with the control group, the W/D value of the rats in the large tidal volume group was obviously increased after mechanical ventilation of 2h, (P0.05), (P0.05). It was said that the severe pulmonary water increased and the pulmonary edema was caused by the mechanical ventilation in the large tidal volume group.
MRNA expression levels of 3.1.3Bmal1, Clock, Per2, Rev-erba and other clock genes
Compared with the free breathing group and the small tidal volume group, the Bmal1, Clock gene mRNA of the lung tissue in the large tidal volume group increased slightly but no significant difference, and the Per2 gene mRNA decreased slightly but had no significant difference. Compared with the free breathing group, the Rev-erb alpha gene mRNA in the small tidal volume group was significantly decreased, and had statistical significance (P0.05), and free call (P0.05). Compared with the low tidal volume group, the Rev-erb alpha gene mRNA in the lung tissue of the large tidal volume group decreased significantly compared with that in the low tidal volume group (P0.05).
Expression level of 3.1.4Rev-erb alpha protein
Compared with the free breathing group, the expression of Rev-erb alpha protein products in the lung tissue of the rats in the small tidal volume group was slightly down, and the expression of the large tidal volume group was significantly down, and was statistically significant (P0.05).
Changes in the concentration of 3.1.5 inflammatory factor TNF- alpha
Compared with the free breathing group, the expression of TNF- alpha in the small tidal volume group was significantly up-regulated, P0.05, and the expression in the tidal volume group was significantly higher than that in the P0.001. group.
The results of the 3.2 second chapters
3.2.1 pathological examination results of lung tissue
The lung tissue of rats in group Control was similar to that of the rats in the large tidal volume group. The lungs were obviously swollen and the surface of the lungs were obviously swollen. The alveolar cavity was visible on the surface. A large number of inflammatory cell infiltration, the thickening of the alveolar wall, the diffuse interstitial hemorrhage and edema were found around the bronchi, and the lungs of the SR9009 rats were slightly visible. The swelling was obvious, but the color and lustre of the surface were basically normal. A small amount of inflammatory cell infiltration and slight pulmonary edema were observed under the light microscope. It was similar to the change of lung tissue in the mechanical ventilation rats of the small tidal volume group. The results showed that SR9009 could significantly reduce the degree of lung injury in the large tidal volume mechanical ventilation rats and effectively improve the inflammatory response of the lung tissue.
The content of TNF- alpha in 3.2.2 lung tissue
Compared with the Control group, the expression level of TNF- alpha in the lung tissue of rats was significantly decreased and there was a statistical significance (P0.05). The results showed that SR9009 could significantly reduce the level of TNF - alpha in lung tissue of rats with large tidal volume and reduce the inflammatory response of lung tissue.
4 Conclusion
(1) the mechanical ventilation caused the injury or necrosis of alveolar epithelial cells and induced the release of inflammatory cytokines in the lung during mechanical ventilation, which led to mechanical ventilation injury. This study successfully simulated the animal model of mechanical ventilation induced lung injury caused by large tidal volume.
(2) lung injury induced by mechanical ventilation may occur in circadian rhythm of lung tissue in rats. Rev-erb alpha plays an important role in lung injury induced by mechanical ventilation in rats.
(3) Rev-erb alpha protein specific agonist SR9009 has a good therapeutic effect on ventilated lung injury. It can significantly reduce the degree of lung injury in the large tidal volume of mechanical ventilation rats, reduce the level of TNF-a caused by the large tidal volume, and reduce the inflammatory response of the lung tissue.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R563.8

【參考文獻】

相關期刊論文 前1條

1 陳彩霞;;上呼吸道感染與生物節(jié)律的關系[J];當代護士(�？瓢�);2009年10期

，

本文編號：2103151