【作者】 张东霞；

【导师】 黄跃生；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 缺血缺氧是严重烧伤早期重要的病理生理现象,而缺血缺氧引发细胞死亡和组织器官损害的根本原因是能量代谢障碍。心脏是一高耗能的器官,线粒体正常的呼吸功能在维持心脏的正常生理活动中发挥重要作用。以往研究已证实,在严重烧伤或心肌缺血缺氧时,线粒体氧化磷酸化关键酶活性降低、ATP合成不足是诱发或加重心肌损伤的重要原因,而心脏作为全身血液循环的动力器官,心肌的损伤和功能减退必将进一步加重全身的缺血缺氧,从而形成恶性循环。线粒体能量代谢障碍是多种复杂因素共同作用的结果,尤其是当机体受到烧伤、创伤、应激、缺氧等刺激时。以往多研究关键酶在能量代谢障碍中的作用,对能量代谢障碍发生发展过程中线粒体的损伤与代偿反应缺乏整体了解,因此,缺少防治线粒体能量代谢障碍的有效干预手段。比较蛋白质组学为我们提供了全貌性了解心肌线粒体在烧伤早期代谢障碍发生发展过程中蛋白质组的差异表达规律,以及在此基础上探索上游分子机制或关键调控环节的手段。本研究利用大鼠烧伤模型,以比较蛋白质组学的方法动态观察烧伤早期心肌线粒体蛋白质组的表达差异,并初步验证了关键的差异表达蛋白活性改变可能引起的生物效应,有助于了解在应激、缺氧等刺激下心肌线粒体的损伤与代偿反应,寻找在心肌线粒体能量代谢障碍的发生发展中起重要作用的关键蛋白分子,为防治烧伤早期心肌损伤提供新靶点与理论依据。一、材料与方法采用动物实验和体外实验相结合的方法1.以Nycodenz密度梯度离心法获取大鼠心肌组织纯化线粒体,并以透射电镜、Western blot法验证其纯度与完整性;2.建立大鼠30% TBSAⅢ°烫伤模型,以正常对照组与烧伤各组纯化的心肌线粒体裂解蛋白行双向凝胶电泳(2-DE)、考马斯亮蓝染色、凝胶扫描后获2-DE图谱,以PDQuest软件分析各组差异表达蛋白点,并将各蛋白点切下行胶内酶解、质谱分析后获差异表达蛋白谱;3.在动物烧伤模型与体外细胞缺氧模型的基础上,验证关键差异蛋白的表达趋势,同时检测以鱼藤酮作用于心肌细胞验证关键蛋白活性受抑后的生物效应,以及心肌线粒体的超微病理损伤。二、主要结果与结论1.采用Nycodenz不连续密度梯度离心法提纯了大鼠心肌线粒体,并以透射电镜与Western blot法证实在不影响线粒体完整性的情况下,提纯过程进一步提高了线粒体的纯度与富集程度;2.以正常对照组与烧伤后1、3小时组的心肌线粒体蛋白行双向电泳,得到聚焦良好、显色清晰、相似性高的2-DE图谱,在各组胶上平均有394.7±5.5、391.0±13.9、385.3±12.6个蛋白点,以PDQuest软件对各组图谱分析后得到10个差异表达蛋白点,将各点行胶内酶解、质谱分析后获得差异表达蛋白谱;3.通过分析差异表达蛋白谱,发现其中8个蛋白在烧伤早期表达下降,分别为:TNF受体相关蛋白1、甲基丁烯酰辅酶A羧化酶1(α)、电子转移黄素蛋白-泛醌氧化还原酶、NADP+依赖的苹果酸酶3、NADH脱氢酶(泛醌)黄素蛋白1、线粒体翻译延伸因子Tu(EF-Tumt)、长链乙酰-辅酶A脱氢酶与短链乙酰-辅酶A脱氢酶;体现了烧伤后的机体损伤,这些蛋白表达降低可能引起的生物效应是ATP合成减少、ROS生成增多、抗氧化能力降低、促进凋亡;另外2个蛋白点表达增高,分别为:推测蛋白LOC498909与ATP合酶F1复合体α亚单位,其意义可能为抵抗上述各种损伤的内源性保护效应;4.在表达下降的蛋白点中我们发现在功能上有密切关系的两个蛋白点,即EF-Tumt与NADH脱氢酶。EF-Tumt为参与NADH脱氢酶蛋白合成的上游因子,而NADH脱氢酶为线粒体呼吸链复合体Ⅰ的催化核心,其下降将引起复合体Ⅰ活性降低,因此,我们推测烧伤后EF-Tumt表达下降可能通过降低NADH脱氢酶的蛋白表达进一步影响呼吸链复合体Ⅰ活性,导致能量代谢障碍;5.为初步证实这一推测,我们首先回复验证了EF-Tumt、NADH脱氢酶亚单位3在各组动物心肌组织中的表达变化,证实了2-DE结果的可靠性;并发现体外培养的缺氧心肌细胞中EF-Tumt蛋白表达亦有降低;在体外以鱼藤酮阻断复合体Ⅰ的生物活性后发现心肌细胞活力下降;同时,以透射电镜观察烧伤大鼠心肌组织发现,烧伤后3小时内,心肌线粒体超微病理损伤逐渐加重,均初步提示EF-Tumt表达下降可能引起的生物效应,但其确切的作用和机制还需进一步验证。更多还原

【Abstract】 Ischemia/hypoxia is one of the key scientific issues following severe burns,which may induce cell death and organic damages for the disturbance of energy metabolism. Myocardium is a highly oxidative tissue that produces more than 90% of its energy from mitochondrial respiration. It has been demonstrated that mitochondria dysfunction in oxidative phosphorylation plays an important role in the inducement and aggravation of myocardial damage during severe burn or following ischemia/hypoxia. As heart is the power organ of circulation, myocardial damages would undoubtedly aggravate ischemia and hypoxia of other organs, resulting in the forming of vicious circle.Disturbance of mitochondrial energy metabolism is the common result of multiple complex factors, especially when the body suffered from burn, trauma, stress or hypoxia. There is little knowledge on the entire mitochondria injury and compensation response during the development of energy metabolism disturbance, so few methods was found effectively in alleviating the disturbance of mitochondrial energy metabolism. Comparative proteomics gives us a global description of the differential expression of myocardial mitochondria proteins in the development of energy metabolism disturbance at the early stage of severe burn, and shed light on the exploration of upstream molecular mechanism, therefore, effective methods alleviating the post-burn energy metabolism disturbance may be found.In this study, comparative proteomics was used to reveal the differential expression of myocardial mitochondria proteins in rats with burn, followed by evaluating the effect induced by changed expression of key proteins, which shed light on the injury and compensation response in myocardial mitochondria when suffered from stress and hypoxia, helping to identify the key protein that playing important roles in the development of energy metabolism disturbance. Those findings will provide new targets and theoretical references in alleviating myocardial injury during early stage after severe burn. I. Materials and methodsBoth in vivo and in vitro experiments were adopted in the present study.1. Purified mitochondria were obtained by Nycodenz density gradient centrifugation, the purity and integrity were identified through electron microscope and western blot analysis.2. A 30% TBSAⅢ°scald rat model was established, 2-DE maps in control and post-burn groups were obtained by comparative proteomics, and then were analyzed with PDQuest software, the differential expression proteins were identified through in-gel digestion and HPLC-chip-MS/MS analysis.3. Both in vivo and in vitro experiments were adopted to verify the expression changes of key protein, and revealed the possible effects through inhibiting the complexⅠactivity by rotenone. At the same time, the ultrastructure damages of myocardial mitochondria were evaluated through electron microscope analysis.II. Results and conclusions1. The purity and integrity of purified myocardial mitochondria isolated by Nycodenz density gradient centrifugation are proved quite well through electron microscope and western blot analysis.2. Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 394.7±5.5, 391.0±13.9, and 385.3±12.6 protein spots were detected from the control, 1 hour post-burn, and 3 hour post-burn groups. All of the maps were analysed with PDQuest software, a total of ten differential expression proteins was successfully identified by HPLC-chip-MS/MS.3. Eight of the ten differential expression proteins show a downregulated expression in post-burn groups, they are TNF receptor-associated protein 1, methylcrotonoyl-Coenzyme A carboxylase 1, acetyl-Coenzyme A dehydrogenase long-chain and short-chain, NADP(+)-dependent malic enzyme 3, ETF-QO, NADH dehydrogenase, EF-Tumt. The decreased expression may result in the disturbance of ATP synthesis, burst of ROS, impairment of the resist ability to ROS injury and apoptosis induction. The protein expression level of LOC498909 and ATP synthaseαsubunit were increased in burn groups, which may be the compensation effect to resist the injury.4. When analyzing the function of differential expression proteins, intrinsic relationship was found between EF-Tumt and NADH dehydrogenase. EF-Tumt is an upstream element that control the protein translation of NADH dehydrogenase, and NADH dehydrogenase is the catalytic core of complex I, so, It can be presumed that EF-Tumt play an important role in the induction of energy metabolism disturbance through inhibiting the activity of complexⅠb y decreasing the protein translation of NADH dehydrogenase.5. To tentatively confirm above presumption, the expressions of EF-Tumt and NADH dehydrogenase subunit 3 were verified in rat myocardium, the results were consistent with those in 2-DE and the reliability of 2-DE was verified; and downregulation of EF-Tumt was found in cultured cardiomyocyte treated with hypoxia. Meanwhile, decreased activity of cardiomyocyte treated with rotenone and aggravated damage of mitochondria ultrastructure in burned rat heart were found. All of the injury may be the effect that induced by the decreased expression of EF-Tumt. However, more studies are needed to conform this effect.更多还原