严重烧伤大鼠肺与膈肌凋亡及其机制的研究

发布时间：2018-03-25 17:37

本文选题：烧伤　切入点：肺　出处：《中国人民解放军医学院》2016年博士论文

【摘要】：目的严重烧伤患者往往伴随呼吸功能障碍,治疗效果差,因此被认为是烧伤早期的主要死因之一。既往研究发现严重烧伤引起的肺损伤和呼吸肌萎缩是造成呼吸功能障碍的两个主要原因。其中,凋亡在烧伤引起的肺损伤中扮演重要角色;而骨骼肌核凋亡也被认为是严重烧伤后四肢骨骼肌萎缩的重要机制之一。因此,本研究拟在此基础上探讨：1、严重烧伤大鼠肺组织凋亡发生的规律、对肺组织的影响，以及Hippo信号通路可能的参与调节机制;2、严重烧伤大鼠膈肌核凋亡发生的规律和死亡受体通路在其中扮演的角色。方法1、Wistar大鼠32只(雄性,清洁级)、,用随机数表法分为假伤组(SB组)、烧伤组(B组)、烧伤12周康复组(12WR组)及烧伤12周康复+二次烧伤组(12WB组)各8只。肺组织标本采集时间为：SB组和B组于致伤后第4天;12WR组和12WB组则为接受为期12周的康复期后,再次分别接受第二次假伤和烧伤,并于伤后第4天搜集标本,方法同前。使用Western印迹法检测肺组织Bc1-2、Bc、-x、Bax,剪切体Caspase-3、YAP、P-YAP、MST、P-MST、 LATS1、LATS2表达水平，使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)检测肺组织细胞凋亡水平,免疫组化检测肺组织P-YAP表达水平，，Masson三色染色和天狼星红苦味酸染色法用于检测肺组织胶原表达水平：2、80只Wistar大鼠(雄性,清洁级)随机分2组：假伤组(n=40)和烧伤组(n=40),并分别在伤后第0、1、4、7、10天每组每次8只大鼠,取腹主动脉血和完整膈肌。使用投射电镜(TEM)观察膈肌凋亡超微结构;使用免疫组化观察膈肌Caspase-3表达;使用酶联免疫吸附试验(ELISA)检测血清sFas、 sFasL及sTRAIL水平;使用Western blot检测膈肌Akt、P-Akt、Bax、Bcl-2、 caspase-8、caspase-3蛋白表达水平。结果1、Masson三色染色和天狼星红苦味酸染色病理均显示12WR组和12WB组肺组织纤维化明显。大鼠肺组织细胞TUN EL凋亡率：B组、12R组及12WB组均显著高于SB组(均P0.05)。B组、12R组及12WB组的剪切体Caspase-3、 P-YAP/YAP、Bax/Bcl-2、P-MST、MST和LATS1均显著高于SB组(均P0.05)。P-YAP免疫组化提示,B组、12R组及12WB组的P-YAP表达增高。2、严重烧伤后大鼠体重明显降低,膈肌重量和CSA也显著下降;透射电镜(TEM)观察到烧伤后膈肌细胞胞质浓缩,胞膜边缘起泡,线粒体肿胀等典型凋亡现象;烧伤组TUN EL阳性细胞率高于假伤组;烧伤组血清sFasL/sFas和sTRAIL高于假伤组(均P0.05);Western Blot结果,烧伤组第1、4天p-Akt明显下降,Bax/Bc1-2于第1、4、7天明显上升,剪切体Caspas-8在第1、4天表达明显增加，剪切体Caspase-3表达水平在第4天达到顶峰;免疫组化Caspase-3提示烧伤后Caspase-3主要在膈肌肌核内表达。结论1、严重烧伤可引起大鼠肺组织出现凋亡,大鼠肺组织Hippo信号通路及下游的YAP蛋白变化规律与凋亡变化相一致,Hippo信号通路可能是调控严重烧伤大鼠肺组织细胞凋亡的重要机制之一；凋亡促进了烧伤后肺纤维化的形成和发展,线粒体凋亡通路可能是其中机制之一。2、严重烧伤可引起大鼠膈肌萎缩,表现为膈肌重量和肌纤维横截面积的下降;凋亡是膈肌萎缩的重要原因,其中,死亡受体通路是烧伤后膈肌凋亡的重要调节机制。
[Abstract]:ObjectiveSevere burn patients accompanied with respiratory dysfunction, poor treatment effect, so it is considered to be one of the major causes of death in early stage of burn. Previous studies have found that severe burns induced lung damage and respiratory muscle atrophy is caused by two main reasons of respiratory dysfunction. Among them, apoptosis plays an important role in lung injury caused by burn and bones; muscle nuclear apoptosis is considered to be one of the important mechanism of postburn limb skeletal muscle atrophy. Therefore, this study intends to investigate on the basis of: 1, apoptosis of lung tissue of rats with severe burn of effects on lung tissue, and the Hippo signal pathway may be involved in regulating mechanism; 2, nuclear apoptosis of diaphragm in severely burned rats and the occurrence regularity of the death receptor pathway in which to play. Methods 1, 32 Wistar rats (male, clean), and with randomly divided into sham injury group (group SB), burn Group (group B), burn 12 week rehabilitation group (group 12WR) and 12 week rehabilitation + two burn burn group (12WB group) with 8 rats in each time. The lung tissue specimen collection: SB group and B group in fourth days after injury; 12WR group and 12WB group accepted rehabilitation period of 12 after a week, once again received second times the sham injury and burn, and collect specimens, on the fourth day after injury. With the method of detection of lung tissue Bc1-2, Western blotting using Bc, -x, Bax, YAP, P-YAP isoform Caspase-3, MST, P-MST, LATS1, and the expression level of LATS2, using dUTP nick end labeling terminal deoxynucleotidyl transferase mediated assay (TUNEL) level in lung tissue cell apoptosis detection, immunohistochemical detection of lung tissue P-YAP expression level, for the detection of lung collagen expression level Masson staining and Sirius red staining method: 2,80 Wistar rats (male clean grade) were randomly divided into 2 groups: the sham injury group (n=40) and burn Injury group (n=40), and 0,1,4,7,10 days after injury respectively in each group 8 rats, abdominal aorta blood and complete diaphragm. Using transmission electron microscopy (TEM) observation of diaphragmatic apoptosis ultrastructure; using immunohistochemical observation of diaphragmatic muscle Caspase-3 expression; using enzyme-linked immunosorbent assay (ELISA) detection of serum sFas, sFasL and sTRAIL levels; using Western blot to detect diaphragmatic Akt, P-Akt, Bax, Bcl-2, Caspase-8, caspase-3 protein expression levels. Results 1, Masson staining and Sirius red staining pathology showed that group 12WR and group 12WB lung fiber is obvious. The rate of apoptosis of EL TUN cells in lung tissue of rats: B group, 12R group and 12WB group were significantly higher than that of group SB (P0.05).B group, shear body Caspase-3, 12R group and 12WB group P-YAP/YAP, Bax/Bcl-2, P-MST, MST and LATS1 were significantly higher than that of group SB (P0.05).P-YAP immunohistochemistry shows, B group, 12R group and 12WB group the expression of P-YAP. High.2, severe burn rats significantly decreased body weight, diaphragmatic muscle weight and CSA decreased significantly; transmission electron microscopy (TEM) observed after burn diaphragmatic muscle cell cytoplasm condensed, cell membrane edge blistering, swelling of mitochondria typical apoptosis; TUN burn group EL positive cells was higher than that in sham group; sFasL/sFas and sTRAIL burn serum group higher than those in sham group (P0.05); Western Blot, burn group at 1,4 days p-Akt decreased significantly, Bax/Bc1-2 increased significantly in the 1,4,7 days, Caspas-8 expression in shear day 1,4 significantly increased the expression level of Caspase-3 isoform peaked in fourth days; immunohistochemistry Caspase-3 after burn Caspase-3 in diaphragm muscle nucleus expression. Conclusion: 1, severe burns can cause rat lung tissue apoptosis, consistent with changes of YAP protein and apoptosis in lung tissue of rats with Hippo pathway and downstream Hippo signaling pathway may be regulated. One of the important mechanisms of cell apoptosis in pulmonary tissues of rats with severe burns; apoptosis promoted the formation and development of pulmonary fibrosis after burn, mitochondrial apoptosis pathway may be one of the mechanisms of.2, severe burns can cause rat diaphragm muscle atrophy, decreased diaphragmatic muscle weight and muscle fiber cross-sectional area; apoptosis is an important reason. Diaphragmatic muscle atrophy is the death receptor pathway after burn diaphragmatic apoptosis an important regulatory mechanism.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R644

【相似文献】