核内受体Rev-erbα在机械通气肺损伤中的作用研究

发布时间：2018-07-06 14:50

本文选题：机械通气 + 肺损伤　；参考：《南方医科大学》2014年硕士论文

【摘要】：1研究背景和目的机械通气(MV)已成为救治呼吸衰竭的重要手段之一,但其本身也可诱发或加重原有的肺损伤,导致机械通气肺损伤(VILI)。VILI的组织病理学改变和其他原因引起的肺损伤相似,均伴有肺泡上皮和血管内皮的广泛性破坏、肺泡毛细血管膜通透性增加、肺水肿、出血、透明膜形成和炎性细胞浸润等病理学改变。自1974年,webb等人最早提出机械通气能导致肺损伤这一概念至今[2], VILI日益受到人们的关注,近年来对VILI的发病机制进行了大量的研究。目前认为VILI主要是由于高气道峰压和大潮气量通气导致吸气末肺组织过度扩张,以及不张的终末小气道或肺泡随机械通气周期性开放和关闭所引起的机械性肺损伤,此外,在机械性损伤的基础上,肺内炎症细胞聚集、活化和释放炎性介质可进一步促进VILI的发生和发展。根据VILI的损伤类型,一般将VILI大致分为以下几种类型,即气压伤、容量伤、不张伤和生物伤。其中前两者属于机械性损伤,一般早期首先出现机械性损伤,随后以炎症细胞、细胞因子介导的生物伤为主,两者相互联系,生物伤作用重要,机制复杂,成为VILI的研究热点。大量研究表明,机械通气产生的异常增高的牵张、剪切力等机械刺激作用于肺细胞,导致肺细胞内众多信号转导信号激活如MAPK通路、NF-κB系统以及细胞膜表面的牵拉敏感性离子通道,使各种炎性细胞如TNF-α IL-1β、IL-8等表达增多引起白细胞向肺组织浸润,这些炎症细胞和细胞因子相互作用,构成一个庞大且复杂的网络体系,不但能直接导致肺组织损伤,还可通过血液循环释放到全身各个组织器官,引起多器官功能障碍(MODS)甚至危及生命。虽然在过去几十年内对VILI的发病机制有了大量的研究也取得了显著的成果,但是其确切机制目前并不是很清楚。最近有人提出机械通气肺损伤可能存在昼夜节律性,这为VILI的机制研究开辟了一条新的道路。昼夜节律是所有生物体对可预测的环境改变的一种综合性的适应,它是可以持续运行并且以大约24h为周期的生物节律,是生物界最普遍的一种生物节律,与许多生理和行为过程有关,如睡眠-觉醒周期、激素水平、体温、血压、呼吸等。昼夜节律的中枢在下丘脑视交叉上核,也存在于机体的各个组织器官,肺组织也不例外。研究显示,人体正常肺功能具有明显的昼夜节律性,潮气量、分钟通气量、平均吸气速率以及呼吸效率等均存在昼夜节律性变化,时钟基因在正常肺组织中呈规律表达以调节正常肺功能。更有研究表明,一些呼吸系统疾病如呼吸道感染、哮喘、非小细胞肺癌等同样也具有一定的昼夜节律性。那么,作为呼吸系统相关疾病的VILI是否也具有昼夜节律性呢?目前国内外鲜有报道。因此,本研究主要从昼夜节律这个新视角来探讨VILI的发病机制,以期为VILI的防治提供一种新思路和药物作用新靶点。 2材料和方法 2.1第一章：机械通气肺损伤大鼠肺组织中核内受体Rev-erba表达改变清洁级同批SD大鼠,体重180-220g,24只,由中山大学实验动物中心提供。随机分为3组(n=8)：F组(自由呼吸组)、LV组(小潮气量组)、HV组(大潮气量组),在光照-黑暗(Light-dark, LD)交替条件下饲养,动物房温维持在20±2℃。采用LD光制(12h：12h)即12小时光照(光照期08：00-20：00)与12小时黑暗(黑暗期20:00-08：00)交替,适应性饲养2周后开始实验。所有大鼠均用10%的水合氯醛0.35g/100g腹腔注射麻醉,仰卧位固定,行右颈总动脉、尾静脉置管术,右颈总动脉置管。待大鼠对口咽部刺激无反应后经口直视下插入14号静脉套管针作为气管导管,血压稳定后,从尾静脉缓慢推入维库溴胺(0.2mg/ml,生理盐水稀释)0.6mg/kg,自主呼吸消失后,连接小动物呼吸机行机械通气。其中F组插管后不给肌松药不行机械通气让大鼠自主呼吸,LV组给予10ml/kg小潮气量行机械通气,HV组给予40ml/kg大潮气量行机械通气,通气时间为2h,机械通气过程中维持泵入维库溴胺(0.2mg/ml)20ml/kg/h。通气完毕后,待自主呼吸恢复平稳即停止通气,拔除气管导管以及动静脉套管针,动脉结扎止血,静脉压迫止血,肌肉和皮肤对应缝合,置大鼠于动物房中单笼饲养。24小时后取大鼠肺组织检测各组肺湿干重比值；HE染色观察各组肺组织病理改变；RT-PCR和Western blot方法检测Bmal1、Clock、Per2、 Rev-erba等时钟基因mRNA和蛋白表达水平。 2.2第二章：特异激动剂SR9009对大潮气量机械通气肺损伤大鼠的治疗效果清洁级同批SD大鼠,体重180-220g,16只,由中山大学实验动物中心提供。随机分为2组(n=8):control组、SR9009组,在光照-黑暗(Light-dark, LD)交替条件下饲养,动物房温维持在20±2℃。采用LD光制(12h：12h)即12小时光照(光照期08：00-20：00)与12小时黑暗(黑暗期20：00-08：00)交替,适应性饲养2周后开始实验。其中SR9009组在麻醉前腹腔注射50mg/kg SR9009,control组注射等量稀释液DMSO,30min后两组大鼠均用10%的水合氯醛0.35g/100g腹腔注射麻醉,仰卧位固定,行右颈总动脉、尾静脉置管术,右颈总动脉置管。待大鼠对口咽部刺激无反应后经口直视下插入14号静脉套管针作为气管导管,血压稳定后,从尾静脉缓慢推入维库溴胺(0.2mg/ml,生理盐水稀释)0.6mg/kg,自主呼吸消失后,连接小动物呼吸机行机械通气。两组均采用大潮气量机械通气,呼吸机参数设置为：VT=40ml/kg,f=40次／分,I：E=1：1.5,通气时间为2h,机械通气过程中维持泵入维库溴胺(0.2mg/ml)20ml/kg/h。通气完毕后,待自主呼吸恢复平稳即停止通气,拔除气管导管以及动静脉套管针,动脉结扎止血,静脉压迫止血,肌肉和皮肤对应缝合,置大鼠于动物房中单笼饲养。24小时后取大鼠肺组织检测各组肺湿干重比值；HE染色观察两组肺组织病理改变；Elisa实验对比两组肺组织TNF—α含量变化。 3结果 3.1第一章结果 3.1.1大鼠肺组织病理改变肺组织病理结果显示,自由呼吸组大鼠肺脏外观正常,光镜下可见肺组织结构完整、肺泡腔清晰、肺泡间质无水肿,无炎性浸润改变；小潮气量组大鼠肺脏外观略显肿胀,但表面色泽基本正常,光镜下可见轻度肺间质水肿和少量巨噬细胞、淋巴和单核细胞等炎症细胞浸润；大潮气量组大鼠肺脏外观明显肿胀,表面可见点状出血,光镜下可见弥漫性肺间质水肿,肺泡腔、血管旁和支气管周围有大量炎性细胞浸润,肺泡间隔明显增厚。 3.1.2肺湿干重比值肺湿干重比值(W/D值)是评价肺水肿的指标,与对照组比较,小潮气量组大鼠肺组织W/D值略微增加；与对照组、小潮气量两组相比,大潮气量组大鼠机械通气2h后,W/D值明显增加,(P0.05),说明大潮气量组机械通气后造成严重肺水增多,肺水肿。 3.1.3Bmal1、Clock、Per2、Rev-erba等时钟基因mRNA表达水平与自由呼吸组和小潮气量组相比,大潮气量组大鼠肺组织Bmal1、Clock基因mRNA略微升高但无显著性差异；Per2基因mRNA略微下降但无显著性差异。与自由呼吸组相比,小潮气量组Rev-erbα基因mRNA呈显著性下降,且有统计学意义(P0.05),与自由呼吸组和小潮气量组相比,大潮气量组大鼠肺组织Rev-erbα基因mRNA呈显著性下降,且有统计学意义(P0.05)。 3.1.4Rev-erbα蛋白表达水平与自由呼吸组相比,小潮气量组大鼠肺组织Rev-erbα蛋白产物表达轻微下调,大潮气量组表达显著下调,且有统计学意义(P0.05)。 3.1.5炎症因子TNF-α浓度变化与自由呼吸组相比,小潮气量组大鼠肺组织TNF-α表达显著上调,P0.05；大潮气量组表达显著上调,P0.001。 3.2第二章结果 3.2.1肺组织病理学检测结果 Control组大鼠肺组织与大潮气量组机械通气大鼠肺组织改变相似,大鼠肺脏外观明显肿胀,表面可见点状出血,光镜下可见肺泡腔、血管旁、支气管周围有大量炎性细胞浸润,肺泡壁增厚,弥漫性肺间质出血和水肿；SR9009组大鼠肺脏外观略显肿胀,但表面色泽基本正常,光镜下可见少量炎症细胞浸润和轻微肺水肿,与小潮气量组机械通气大鼠肺组织改变相似。该结果说明SR9009能明显减轻大潮气量机械通气大鼠肺损伤程度并有效改善肺组织炎症反应。 3.2.2肺组织TNF-α含量与Control组比较,大鼠肺组织TNF-α表达水平明显降低,且有统计学意义(P0.05)。该结果说明SR9009能明显降低大潮气量机械通气大鼠肺组织肿瘤坏死因子α水平,减轻肺组织炎症反应。 4结论 (1)大潮气量机械通气时由于机械牵拉导致肺泡上皮细胞损伤或坏死,并诱导肺内炎性细胞因子的释放,从而导致机械通气肺损伤,本研究成功模拟了大潮气量所致机械通气肺损伤动物模型； (2)大鼠机械通气所致肺损伤可能发生肺组织昼夜节律紊乱；Rev-erbα在大鼠机械通气肺损伤中发挥重要作用； (3) Rev-erbα蛋白特异激动剂SR9009对机械通气肺损伤具有良好的治疗效果,能显著减轻大潮气量机械通气大鼠肺损伤程度,降低大潮气量所致TNF-a水平,减轻肺组织炎症反应。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R563.8

【参考文献】