【作者】 王俊锋；

【导师】 刘长庭；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年医学呼吸病学， 2009， 博士

【摘要】 目的:失重会使肺功能受到影响,并可导致一定程度肺组织损伤。本实验采用大鼠尾悬吊模拟失重的方法,研究模拟失重后大鼠肺组织蛋白组学变化情况,深入了解失重所致肺损伤的发生机制;同时选择N-乙酰半胱氨酸和银杏叶提取物进行药物干预,观察大鼠肺组织细胞凋亡和一氧化氮表达情况的变化,为失重所致肺损伤的药物防护提供理论依据。方法:1.蛋白组学研究:选择健康雄性S-D大鼠20只,体重180-220g,随机分为模拟失重7天和正常对照2组,采用尾悬吊-30°作为模拟失重模型。尾悬吊结束时,用10%水合氯醛(30ml/kg)腹腔注射麻醉后,打开胸腔取出肺组织并提取肺组织蛋白,蛋白提取后采用双向电泳技术(2-DE)建立差异表达图谱,通过凝胶成像分析和人工比对,确定差异蛋白质斑点,然后对每个差异蛋白点进行胶内酶切,采用HDMS进行液相串联质谱(LC-MS/MS)分析结合数据库检索鉴定蛋白质。最后对差异蛋白的生物学功能进行较为详细的了解和分类,进一步深入研究它们的功能。2.药物干预研究:选择健康雄性S-D大鼠40只,体重180-220g,随机分为模拟失重7天、正常对照、N-乙酰半胱氨酸干预和银杏叶提取物干预4组,采用尾悬吊-30°作为模拟失重模型。尾悬吊结束时,用10%水合氯醛(30ml/kg)腹腔注射麻醉后,打开胸腔取出肺组织制作石蜡切片。采用原位细胞凋亡检测法、免疫组织化学法和原位杂交法分别观察大鼠肺组织细胞凋亡、诱导型一氧化氮合酶(iNOS)和bcl-2、bax及iNOS mRNA的变化。结果:1.蛋白组学变化:应用2-DE对悬吊组和对照组大鼠肺组织样品进行分离和对比,建立差异表达图谱,通过凝胶成像分析和人工比对,确定差异蛋白质斑点17个,其中13个上调,3个下调,1个消失。这些点分布在等电点4.65-8.61、分子量15972-60317范围内,最大表达上调6.9倍,下调最低至0.1倍。经胶内酶切和质谱鉴定,共鉴定出17种蛋白质,其中13种上调,3种下调,1种消失,与细胞代谢相关的5个,与细胞功能相关的4个,与细胞结构蛋白相关的3个,与蛋白降解系统相关的2个,与信号传导相关的3个。它们可能与细胞能量代谢、应激与炎症反应、细胞损伤与修复、细胞内信号转导和其它细胞功能活动有关,在细胞免疫应答、细胞凋亡等方面起着重要的作用。2.药物干预结果:悬吊组大鼠肺组织细胞凋亡指数(23.1%±2.6%)明显高于正常对照组(2.2%±1.1%)(P＜0.05),而N-乙酰半胱氨酸干预组大鼠肺组织细胞凋亡指数(7.5%±1.6%)显著低于尾悬吊组(P＜0.05),大鼠肺组织bcl-2/baxmRNA的表达也有相应的变化(P＜0.05)。悬吊组大鼠肺组织iNOS表达水平明显高于对照组(P＜0.05),而N-乙酰半胱氨酸干预组大鼠肺组织iNOS表达水平明显低于7天悬吊组(P＜0.05),大鼠肺组织iNOS mRNA表达也有相应的变化(P＜0.05)。同样,银杏叶提取物干预组大鼠肺组织细胞凋亡指数(8.1%±1.7%)显著低于尾悬吊组(P＜0.05),其bcl-2/baxmRNA的表达也有相应的变化(P＜0.05)。而且银杏叶提取物干预组大鼠肺组织iNOS表达水平也明显低于7天悬吊组(P＜0.05),其iNOS mRNA表达也有相应的变化(P＜0.05)。结论:模拟失重后大鼠肺组织蛋白组学发生变化,有些蛋白表达上调,而有些蛋白表达下调甚至消失,影响细胞能量代谢、应激与炎症反应、细胞损伤与修复以及细胞内信号转导等细胞功能活动,在失重所致肺组织损伤中发挥着重要的作用。模拟失重时大鼠肺组织细胞凋亡水平增高,iNOS表达水平也增高,而N-乙酰半胱氨酸和银杏叶提取物均可抑制模拟失重大鼠肺组织的细胞凋亡过度,并能降低iNOS的表达水平。更多还原

【Abstract】 ObjectiveLung function changes during weightlessness,and a certain degree pulmonary tissue injury comes out.Tail-suspension was used as weightlessness simulation to study proteomic changes of pulmonary tissues in rats during simulated weightlessness,so as to investigate the mechanism of lung injury caused by weightlessness.And the changes of cell apoptosis and nitric oxide in pulmonary tissues in rats during simulated weightlessness were examined after N-acetylcysteine and ginkgo biloba extract were used to treat respectively,so as to supply the theoretical foundation of medicine protection of lung injury caused by weightlessness.Methods1.proteomic analysis:20 Sprague-Dawley male rats(weight range,180 to 220 g) were randomly divided into 2 groups:tail-suspension for 7 day group,control group. Tail-suspension(-30°) was used to simulate the physiological effects of weightlessness. The pulmonary tissues in rats were taken out after the rats were anaesthetized by abdominal injection with the 10%hydration chloraldehyde(30ml/kg) when the suspension ended.Using two-dimensional electrophoresis(2-DE) the different expression map was established after the proteins were extracted from pulmonary tissues.And proteins express differently was determined by comparing the level of each dots with gel imaging software and manually.Digest in gel was performed first and proteins were identified by high definition mass spectrometry(HDMS) combining with database.Finally,the function of these identified proteins was known and classified.2.drug intervention:40 Sprague-Dawley male rats(weight range,180 to 220 g) were randomly divided into 4 groups:tail-suspension for 7 day group,control group, N-acetylcysteine treatment group and ginkgo biloba extract treatment group. Tail-suspension(-30°) was used to simulate the physiological effects of weightlessness. The rats were anaesthetized by abdominal injection with the 10%hydration chloraldehyde (30ml/kg) when the suspension ended.The pulmonary tissues of rats were cut into paraffin sections.The condition of cell apoptosis in pulmonary tissues was detected with in situ death detection,the expression of inducible nitric oxide synthase(iNOS) was detected with immunohistochemistry and the expression of bcl-2,bax and iNOS mRNA were detected by hybridization in situ.Results1.proteomic changes:The different expression map was established by using two-dimensional electrophoresis(2-DE) to separate the pulmonary tissue extract in rats of tail-suspension group and control group.17 differential expression proteins were determined by comparing the level of each dots with gel imaging software and manually, in which.13 differential expression proteins were up-regulated,3 differential expression proteins were down-regulated and 1 differential expression proteins disappeared.All were in the region of molecular masses of 15972-60317 and an isoelectric point of 4.65-8.61 respectively.Among them,the maximum and minimus change was 6.9 folds and 0.1 folds. 17 proteins were identified by HDMS and in which.13 proteins were up-regulated,3 proteins were down-regulated,1 protein disappeared.The identified proteins included cytoskeletal proteins,degradation system related proteins,proteins involved in signal transduction,metabolism and other cell function.They may be related to cell energy metabolism,stress and inflammatory response,cell injury and repair,intracellular signal transduction and other cell function,and play an important role in cell-mediated immune response,apoptosis,etc.2.drug intervention:The index of cell apoptosis in pulmonary tissues of rats suspended for 7 days was higher significantly than that of control group(P＜0.05),the index of cell apoptosis in pulmonary tissues of rats in N-acetylcysteine treatment group was lower significantly than that of tail-suspension for 7 days group(P＜0.05).And the expressions of bcl-2/bax mRNA in pulmonary tissues had the corresponding changes.The level of iNOS in pulmonary tissues of rats suspended for 7 days was higher significantly than that of control group(P＜0.05),the level of iNOS in pulmonary tissues of rats in N-acetylcysteine treatment group was lower significantly than that of tail-suspension for 7 days group(P＜0.05).And the expressions of iNOS mRNA in pulmonary tissues had the corresponding changes.At the same,the index of cell apoptosis in pulmonary tissues of rats suspended for 7 days was higher significantly than that of control group(P＜0.05),and the index of cell apoptosis in pulmonary tissues of rats in ginkgo biloba extract treatment group was lower significantly than that of tail-suspension for 7 days group(P＜0.05).And the expressions of bcl-2/bax mRNA in pulmonary tissues had the corresponding changes. The level of iNOS in pulmonary tissues of rats suspended for 7 days was higher significantly than that of control group(P＜0.05),the level of iNOS in pulmonary tissues of rats in ginkgo biloba extract treatment group was lower significantly than that of tail-suspension for 7 days group(P＜0.05).And the expressions of iNOS mRNA in pulmonary tissues had the corresponding changes.ConclusionThere were proteomic changes of pulmonary tissues in rats during simulated weightlessness,in which some proteins were up-regulated while some proteins were down-regulated,even disappeared.They may be related to cell energy metabolism,stress and inflammatory response,cell injury and repair,intracellular signal transduction and other cell function,and play an important role in the mechanism of lung injury caused by weightlessness.The levels of cell apoptotic and iNOS expression in pulmonary tissues of rats increased during simulated weightlessness.N-acetylcysteine and ginkgo biloba extract could suppress cell apoptosis and iNOS expression in pulmonary tissues of rats during simulated weightlessness.更多还原