摘要
背景:肺缺血再灌注损伤过程中肺组织细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3表达的动态变化及其可能的作用机制有待观察。目的:观察大鼠肺缺血再灌注损伤中肺组织细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶3表达的动态变化,并分析细胞凋亡在肺缺血再灌注损伤中作用及可能机制。设计:随机对照动物实验。单位:南京医科大学附属南京第一医院急诊中心。材料:实验于2006-04/2006-09在南京医科大学附属南京第一医院动物实验室及南京市放射免疫检测中心完成。选用28只雄性健康清洁级SD大鼠,体质量250￣350g,鼠龄49￣76d,由南京医科大学实验动物中心提供。随机数字表法将大鼠分为实验组及对照组,每组14只。方法:①实验干预:实验组大鼠参照Eppinger等的方法进行建立肺缺血再灌注模型,大鼠给予阻断肺门45min(观察肺无舒缩为阻断标准),再开放3,6h(再灌注以肺恢复舒缩为标准)后取材,每次取7只,对照组仪行开胸术,于相同时间点同样方法分离肺组织。②实验评估:采用Annexin-V-PI双染法通过流式细胞仪检测肺组织凋亡细胞,计算凋亡率;采用免疫组织化学法及图像分析法观察肺脏天冬氨酸特异性半胱氨酸蛋白酶3的表达;计算2组大鼠左肺湿干质量比;计算肺泡损伤数比值进行肺组织损伤定量评价;光镜下苏木精-伊红染色观察肺脏的病理形态学变化。主要观察指标:①肺组织细胞凋亡率及天冬氨酸特异性半胱氨酸蛋白酶3的表达。②肺湿干质量比及肺组织损伤定量评价结果。③肺组织病理形态学变化。结果:纳入大鼠28只均进入结果分析,无脱落。①实验组肺缺血再灌注3,6h肺组织细胞凋亡率较对照组有明显增加,差异有统计学意义(P<0.01),实验组缺血再灌注6h凋亡率较3h有所降低(P<0.05)。②实验组大鼠肺组织天冬氨酸特异性半胱氨酸蛋白酶3表达3h达到最高,6h稍有下降,较对照组各时间点表达增加(P<0.01)。③实验组肺缺血再灌注3,6h肺泡损伤数比值及肺脏湿干质量比值较对照组均明显增加(P<0.01),实验组缺血再灌注6h两比值较3h有所增加(P<0.05)。④实验组肺泡结构破坏、塌陷、消失,肺泡间隔有大量的炎性细胞浸润,肺缺血再灌注6h较3h更严重。对照组无明显改变。结论:肺缺血再灌注早期可能通过天冬氨酸特异性半胱氨酸蛋白酶3表达的改变,激活细胞的凋亡途径,诱导肺组织细胞凋亡的发生,从而导致肺损伤的发生。
BACKGROUND： The dynamic changes of pneumocyte apoptosis and aspartate-specific cysteine proteases-3 （caspase-3） expression in lung tissue of rats during the process of lung ischemia/reperfusion （I/R） injury and the possible action mechanisms remain unclear. OB3ECTIVE： This study was to observe the dynamic changes of pneumocyte apoptosis and caspase-3 expression in the rat lung tissue during the process of lung I/R injury, and to analyze the role of pneumocyte apoptosis and the possible action mechanism. DESIGN ： A randomized controlled animal experiment SEI-rING： Emergency Center, First Hospital, Nanjing Medical University MATERIALS： This study was carried out in the Animal Laboratory of the First Hospital of Nanjing Medcial University and Nanjing Center for Radioimmunity between April 2006 and September 2006. Twenty-eight male healthy SD rats of clean grade, with body weight of 250 to 350 g, aged 49 to 76 days, were provided by the Experimental Animal Center of Nanjing Medical University. The involved rats were randomized into experimental group and control group, with 14 rats in each. METHODS： （1） Experimental intervention： Rats in the experimental group were created into models of lung I/R injury according to the method of Eppinger et al. They were occluded for 45 minutes at the porta of lung （no systolic and diastolic reactions in lung tissue being considered as successful occlusion）, and then they were reperfused （recovery of systolic and diastolic function being considered as successful reperfusion）; After that, lung tissues were harvested at 3 and 6 hours after lung I/R injury, 7 rats at each time point. Each rat in the control group was subjected to a thoracotony only, but lung tissues were isolated at the same time point by the same method. （2）Experimental evaluation： Apoptotic cells in the lung tissue were detected with a flow cytometer by Annexin-V-PI staining, and apoptosis rate was calculated. Caspase-3 expression in the tung tissue was observed by immunohistochemical method and image analysis. Wet to dry weight ratio（W/D） of lung tissue of rats in the two groups was calculated; the number of injured pulmonary alveoli at I/R 3 hours/that at I/R 6 hours was calculated for quantitative evaluation of injured lung tissue; Patho-morphological changes of lung tissue were observed by haematoxylin ＆ eosin staining under an optical microscope. MATN OUTCOME MEASURES： （1）Pneumocyte apoptosis rate and caspase-3 expression in the lung tissue. （2）W/D of lung tissue and quantitative evaluation of injured lung tissue. （3） Patho-morphological changes of lung tissue. RESULTS： Twenty-eight rats were involved in the final analysis, without deletion. （1）Pneumocyte apoptosis rates in the experimental group at I/R 3 and 6 hours were significantly increased as compared with control group （P 〈 0.01）. In the experimental group, pneumocyte apoptosis rate was decreased a little at I/R 6 hours than at I/R 3 hours （P 〈 0.05）. Caspase-3 expression in the lung tissue of rats of experimental group reached its top at I/R 3 hours, and was decreased a little at I/R 6 hours. At each time point, caspase-3 expression in the experimental group was increased as compared with control group （P 〈 0.01）. （3） In the experimental group, the number of injured pulmonary alveoli at I/R 3 hours/that at I/R 6 hours and W/D ratios of lung tissues were significantly increased as compared with control group （P 〈 0.01）. In the experimental group, two ratios at I/R 6 hours were higher than those at I/R 3 hours （P〈 0.05）. （4） In the experimental group, the structure of pulmonary alveoli was destructed, collapsed and disappeared; lots of inflammatory cell infiltration was found; Patho-morphological changes of injured lung tissue at I/R 6 hours were severer than those at I/R 3 hours. No obvious changes were found in the control group. CONCLUSTON： At the early stage of lung I/R injury, the alteration of caspase-3 maybe activate pneumocyte apoptosis and induce the apoptosis of lung tissue, and thereby leads to lung injury.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第43期8792-8795,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
南京市医学科技发展项目(YKK06063)
南京市科技发展计划项目(200601056)~~
