【作者】 李峰；

【导师】 郭振荣；

【作者基本信息】 中国人民解放军军医进修学院 ， 烧伤外科学， 2003， 博士

【摘要】 严重烧伤后由于皮肤屏障的破坏、休克、感染及焦痂毒素等刺激因素，使交感神经系统兴奋性增强，多种体液介质大量的释放，使机体代谢率大幅升高，常处于高代谢状态。长期的高代谢状态必将削弱患者抵抗力，易发生各种并发症。抑制高代谢反应的发生强度、缩短其发生过程是临床治疗的重要方面。解偶联蛋白即是这样一类蛋白：定位于线粒体内膜，多在对能量的产生、储存有重要影响的骨骼肌、肝脏、脂肪组织表达，主要作用是降低线粒体内膜两侧的质子梯度，造成糖、脂肪等能量代谢底物的氧化与ADP磷酸化过程解偶联，能量不是被储存在高能磷酸键中，而是以热能形式散发。以此方式散失的热量可占基础代谢的相当比例。目前尚未出现某种药物可有效地将代谢率调整到较适宜的水平。对烧伤病人而言，虽然伤后病理生理变化过程十分复杂，但焦痂的存在无疑是导致这些病理生理变化过程和代谢率改变的主要因素。积极主动的切除焦痂、覆盖创面，争取缩短创面愈合时间是烧伤救治的主要措施，同时也可使代谢率有效地降低。 实验目的 临床实践证明，休克期切痂在提高救治水平及抑制高代谢率等方面，比传统的伤后4-7天切痂效果更加显著。在以往的研究中可观察到实施休克期切痂的患者或实验动物，术后代谢率显著下降，说明伤后坏死组织和创面的存在不但是影响患者生存，也是导致高代谢率的关键因素。随着科研手段的不断进步，有必要从分子水平上考察烧伤后代谢率升高及切痂可抑制代谢率升高的机理。本实验研究首先观察了30％Ⅲ度烫伤大鼠伤后、及实施休克期切痂或常规延迟切痂术后血清中与高代谢关系密切的激素、细胞因子，和骨骼肌、肝脏、脂肪组织解偶联蛋白表达的改变；然后在离体条件下观察焦痂提取物对骨骼肌细胞解偶联蛋白表达的影响，从代谢的角度论证休克期切痂的必要性。 本实验研究的目的在于： l观察大鼠烫伤后及伤后不同时间切痴对血清一些激素、细胞因子及 瘦素、游离脂肪酸谱和血脂的影响； 二观察大鼠烫伤后及伤后不同时间切痴对骨骼肌、肝脏、白色脂肪组 织 UCP21：TI＝－xriA。UCP3 mKNA表达的影响；对骨骼肌、肝脏能量代谢的 影响； 3体外培养大鼠骨骼肌细胞，制备焦痴提取物，观察焦痴提取物对骨 骼肌细胞UCPZmRNA、UCP3InRNA表达的影响。 材料与方法 1．动物模型：以30％TBSAlll度烫伤大鼠为模型，分4组：A组（烫伤对 照组）分别于伤后8、24、96、120、168h活杀；B组（伤后sh切痴）于伤 后抡、孤、U几1“h活杀；C组（伤后2虬切咖于伤后96、1m、1朋h 活杀；D组（伤后96h切痴）于伤后120、168h活杀。各时相点均设一组动 物，另设一组正常对照组作为伤前对照，每组8只大鼠。B、C．D各组分 别于伤后8、24、96h切痴后植以大张异体皮，邮包包扎。留取血清、骨 骼肌、肝脏、附睾脂肪垫。 2．检测指标： 门）血清皮质醇、胰高血糖素、胰岛素、血糖、血脂、游离脂肪酸谱 水平改变规律； C）血清瘦素、脂肪组织OBmRNA表达改变规律； 厂阻清 TN’F a、肝脏 TMi a InRNA表达改变规律； （4）RT－PCR测定骨骼肌UCPZmRNA、UCP3mRNA，脂肪组织、肝脏 UCPZInRNA水平表达改变规律；肝脏、骨骼肌ATP、能荷改变规律； Western blotting测定骨骼肌 UCPZ蛋白表达规律；兔疫组化观察骨骼肌、 肝脏、脂肪组织UCPZ蛋白表达规律； 3体外实验部分，通过培养大鼠骨骼肌细胞并制备了烫伤大鼠焦痴提 取物，RT－PCR ？k测定骨骼肌细胞培养液中加入焦痴提取物后不同时间 UCPZInRNA、UCP3mRNA表达改变。 结果 1烧伤后血清皮质醇、胰高血糖素、血糖水平显著升高；胰岛素水平 3 先升后降；血清游离脂肪酸水平在伤后存在小幅下降而后升高；休克期切 痴可有效抑制脂动员，使异常各指标较快地接近正常。 2、烧伤后血清瘦素、脂肪组织OBmRNA表达均显著降低，实施休克 期切痴可使其表达水平较快恢复正常。 3烧伤后血清 TWh。、肝脏 TNF a tTlltrvA表达均显著升高；实施休 克期切痴可使其水平较快恢复正常。 4烧伤后骨骼肌UCPZlllrtrvA、UCP3mANA，脂肪组织。肝脏 UCPZmRNA表达均显著上调；骨骼肌、肝脏ATP含量、能荷降低；实施 休克期切哪可使 UCP与 UCP3 InRNA表达水平、ATP含量、能荷较快恢 复正常。 5离体实验表?更多还原

【Abstract】 The metabolic rate will increase after major burns, because excitability of sympathetic nervous system will be reinforced and the amount of infective medium will be raised, which may be resulted from some stimulating factors such as destruction of skin barrier, shock, infection, burned eschar toxins, etc. Hypermetabolism will do harm to the burned patients and many complications will be easy to occur if it lasted for long time. To suppress the intensity as well as shortening the course of hypermetabolism is an important aspect of the treatment. Uncoupling proteins (UCPs), located in the inner membran of mitochondria, express in some tissues, for instance, skeletal muscle, liver and white adipose tissue, which have certain influences on the yield and storage of energy. UCPs could uncouple oxidation of substrate and phosphorylation of ADP, so that the energy released from oxidation of carbohydrate and lipid will not be stored in the energy-rich phosphate bond, but distributed as heat. It accounts for a certain protortion in BMR.A specific medicine, which could regulate metabolic rate to a appropriate level, has not been found until now. For the burned patients, although the pathophysiological alterations are complicated, the presence of eschar is undoubtedly one of the key factors inducing the pathological process and alteration of metabolic rate. To conduct escharectomy and cover the wounds actively, try to shorten the progress are the main measures of treatments. At the same time, it could reduce metabolic rate effectively.OBJECTIVEEscharectomy during shock stage has shown pretty good effects in terms of improving medical treatment level as well as suppressing hypermetabolism. In previous studies, it could be found that metabolic rate declined markedly in those patients or animals who received escharectomy during shock stage. It could be classified that existences of necrotic tissues and wounds not only impact survival rate of the patients, but also are key factors inducing hypermetabolism. Along with the progresses of scientific research means, it’s necessary to explore the mechanisms of hypermetabolism postburns and the suppressive effects of escharectomy in molecular level. In the present study, some hormones, cytokines, which are related to hypermetabolsim, and UCPs expression in skeletal muscle, liver and white adipose tissue are investigated inthe 30% third degree scalded rats. In order to further demonstrate the necessity of conducting escharectomy during shock stage, the effects of eschar extraction on expression of UCPs in skeletal muscle cells in vitro were also explored. The objective of the present study are as follows:1. To observe the effects of burn injuries and escharectomy at different time on the level of several hormones, cytokines, leptin and free fatty acid in plasma.2. To observe the effects of burn injuries and escharectomy at different time on expression of UCP2mRNA, UCP3mRNA in skeletal muscle, liver and adipose tissue, as well as energy metabolism in skeletal muscle and liver.3. To culture skeletal muscle cells in vitro and make eschar extraction of scalded rats skin. To observe the effects of eschar extraction on expression of UCPs in skeletal muscles in vitro.METHODS1. Animal: 30% TBSA full thickness scalded rats were employed and divided into four groups. Rats in group A (scalded control) were killed at 8, 24, 96, 120, 168h respectively after burns; rats in group B (escharectomy at 8h after burns) were killed at 24, 96, 120, 168h after burns; rats in group C (escharectomy at 24h after burns) were killed at 96, 120, 168h after burns; rats in group D (escharectomy at 96h after burns) were killed at 120, 168h after burns. There are eight rats in each time point, and eight rats which were not scalded were signed as normal control. Rats in group B, C, D received escharectomy at 8,24,96h respectively. Serum, skeletal muscle, liver, epididymidis fat pad were collected when the rats were killed.2. The parameters analyzed are as follows:更多还原