模拟失重肺组织蛋白组学变化和药物干预对一氧化氮表达及凋亡的影响

发布时间：2018-03-25 15:18

本文选题：大鼠　切入点：失重模拟　出处：《中国人民解放军军医进修学院》2009年博士论文

【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。
[Abstract]:Objective:
Weightlessness can cause lung function affected, and can cause a certain degree of lung injury. In this experiment, using weight loss method of rat tail suspension model, simulated weightlessness in rats after lung tissue proteome changes, in-depth understanding of the mechanism of lung injury induced by weightlessness; at the same time choose N- acetylcysteine and Ginkgo biloba extract drug intervention, observation of rat lung cell apoptosis and expression of nitric oxide changes, and provide a theoretical basis for the drug loss of lung injury induced by protection.
Method:
Study on the 1. protein group: 20 healthy male S-D rats, weighing 180-220g, were randomly divided into 7 days of simulated weightlessness and 2 normal control group, the tail suspended -30 degrees as the simulated weightlessness model. At the end of the tail suspension, with 10% chloral hydrate (30ml/kg) intraperitoneal injection of anesthesia, thoracic cavity open lung tissue and lung tissue protein extraction and two-dimensional electrophoresis using after protein extraction (2-DE) to establish differential expression patterns, by gel imaging analysis and artificial comparison, determine the different protein spots, and then for each different protein spots were in gel digestion, using HDMS liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis combined with the identification of protein database retrieval. Finally the understanding and detailed classification of biological functions of proteins, further study on their function.
Study on the intervention of 2. drugs: 40 healthy male S-D rats, weighing 180-220g, were randomly divided into 7 days of simulated weightlessness, normal control, intervention N- N-acetylcysteine and Ginkgo biloba extracts 4 groups by tail suspension -30 degrees as the simulated weightlessness model. At the end of the tail suspension, with 10% chloral hydrate (30ml/kg) intraperitoneal injection after anesthesia, open the chest to remove the lung tissue paraffin sections. In situ apoptosis assay, cell apoptosis in pulmonary tissues of rats were observed by immunohistochemistry and in situ hybridization, inducible nitric oxide synthase (iNOS) and Bcl-2, Bax and iNOS change mRNA.
Result:
The changes of 1. groups: the application of 2-DE to protein suspension group and control group rat lung tissue samples were separated and compared, the establishment of differential expression patterns, by gel imaging analysis and manual comparison, determine the different protein spots 17, of which 13 up-regulated and 3 down regulated, 1 disappeared. The isoelectric point distribution in 4.65-8.61, the molecular weight range of 15972-60317, the maximum expression of 6.9 times, the lowest down to 0.1 times. After in gel digestion and mass spectrometry, we identified 17 proteins, including 13 up-regulated and 3 down regulated, 1 disappeared, 5 related to cell metabolism, 4 associated with cell function 3, related to cell structure proteins, 2 related protein degradation system, 3 related to signal transduction. They may be cell energy metabolism, stress and inflammatory reaction, cell damage and repair, intracellular signal transduction and other cell function related activities in cells The immune response, cell apoptosis and other aspects play an important role.
2. drug intervention results: the apoptosis index of lung tissue suspension group rats (23.1% + 2.6%) was significantly higher than the normal control group (2.2% + 1.1%) (P < 0.05), while the N- N-acetylcysteine intervention index group rats lung tissue apoptosis (7.5% + 1.6%) was significantly lower than that of the tail suspension group (P < 0.05). The expression of bcl-2/baxmRNA in lung tissue of rats with corresponding changes (P < 0.05). The level was significantly higher than the control group iNOS expression in lung tissue of rats suspension (P < 0.05), while the N- acetylcysteine intervention in lung tissue of iNOS rats was significantly lower than the expression level of the 7 day suspension group (P < 0.05), rats the lung tissue iNOS mRNA expression also has corresponding change (P < 0.05). Similarly, Ginkgo biloba extract intervention index of rats lung tissue apoptosis (8.1% + 1.7%) was significantly lower than that of the tail suspension group (P < 0.05), the expression of bcl-2/baxmRNA also have corresponding change (P < 0.05) and Ginkgo biloba extract intervention group The expression level of iNOS in rat lung tissue was also significantly lower than that in the 7 day suspension group (P < 0.05), and the expression of iNOS mRNA also had a corresponding change (P < 0.05).
Conclusion:
After simulated weightlessness rats lung tissue proteome changes, expression of some proteins, and some protein expression decreased or disappeared, cell energy metabolism, stress and inflammatory reaction, cell damage and repair and intracellular signal transduction cell function activity, plays an important role in the weight loss caused by lung injury. Simulated weightlessness rat lung tissue cell apoptosis increased, the expression level of iNOS increased, while N- acetylcysteine and Ginkgo biloba extract can inhibit the apoptosis of simulated weightlessness rat lung tissue over expression level and lower iNOS.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R852.22

【参考文献】