【作者】 胡清刚；

【导师】 柴家科；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2010， 博士

【摘要】 目的：丙泊酚能通过多个环节有效预防和调节危重烧创伤后应激紊乱、过度炎症反应和免疫失衡及代谢紊乱,提高危重患者的生存率。但近年有文献报道大剂量(> 4mg·kg-1·h-1)长时间(＞48h)输注后可能诱发低发生率但高死亡率的“丙泊酚输注综合征(PRIS)",使其用于重症患者镇静的安全性受到质疑。“横纹肌溶解症(RM)”在PRIS的发生和转归中起重要作用,而泛素-蛋白酶体途径在骨骼肌损伤和异常代谢中起着关键性作用。本课题利用动物模型和体外细胞培养,将小剂量丙泊酚用于烧伤后持续镇静,观察：1.对应激激素、炎症介质释放的影响；2.对血气、血生化及主要器官组织病理学影响；3.对骨骼肌结构及泛素系统基因表达的影响。系统地评估小剂量丙泊酚用于严重烧伤后持续镇静的安全性和有效性,从基因水平探讨对骨骼肌泛素系统的影响,为丙泊酚应用于危重烧伤患者的镇静治疗提供理论依据。材料和方法：1.动物实验(1)24只健康日本大耳白兔随机分为烫伤镇静组、烫伤对照组、假伤镇静组、假伤对照组,烫伤组麻醉后制成30%体表总面积(TBSA)的Ⅲ烫伤,假伤组麻醉后温水假伤,其它处理与烫伤组相同。伤后3h,镇静组给予6.2mg·kg-1·h-1(等效于人体2mg·kg-1·h-1)的1%丙泊酚持续输注48h,对照组给予相同输注速率和容积的生理盐水。取烫伤前、伤后3h、伤后6h(镇静3h)、伤后9h(镇静6h)、伤后51h(镇静48h)各点的血标本；镇静48h后麻醉动物,取心、肺、肝、肾、肠、骨骼肌组织、尿标本。(2)检测血浆主要应激激素、血糖、血清主要细胞因子含量；(3)检测血气、血生化、血和尿肌红蛋白含量；(4)光镜下观察主要脏器及骨骼肌的组织病理学变化,电镜下观察骨骼肌超微结构；(5)测定骨骼肌组织泛素-蛋白酶体相关成分基因表达变化。2.离体细胞实验(1)建立稳定的兔骨骼肌卫星细胞分离、培养、纯化、融合的方法；(2)将丙泊酚(2μg/ml)、地塞米松(DXM、100μmol/ml)、去甲肾上腺素(NE,10-7mmol/L)与分化成熟的骨骼肌肌管共同培养48h,检测肌管泛素-蛋白酶体相关成分基因表达变化。结果：1、与伤前比较,严重烫伤后血浆肾上腺素、促肾上腺皮质激素(ACTH)、胰岛素含量降低(p＜0.05),皮质醇(COR)含量持续升高(p＜0.05),烫伤镇静组COR高于烫伤对照组(p＜0.05),假伤镇静组较假伤对照组ACTH含量降低(p＜0.05)；2、与伤前比较,严重烫伤后血清TNF-α、IL-1、IL-10均升高(p＜0.05),泊酚镇静降低其升高幅度(p＜0.05)；3、与对照组比较,镇静组动脉血二氧化碳分压、氧分压无显著变化(p＞0.05),烫伤镇静组血乳酸、葡萄糖含量较烫伤对照组降低(p＜0.05)；4、丙泊酚镇静可缓解严重烫伤后血清ALT、AST、CK、CK-MB、血和尿肌红蛋白含量的升高(p＜0.05或p＜0.01),对假伤组上述参数无显著影响(p＞0.05),但血胆固醇、甘油三酯升高(p＜0.05)；5、严重烫伤后心、肺、肝、肾、肠、骨骼肌均有不同程度的损伤,丙泊酚镇静可以减轻其损伤,假伤组镇静后无明显改变；6、丙泊酚镇静能显著抑制烫伤后骨骼肌编码泛素、E2-14KDa酶、E3α酶、C2亚基基因的高表达(p＜0.05或p＜0.01),对假伤组无影响(p＞0.05)；7、丙泊酚对肌管泛素系统基因表达无显著影响(p＞0.05),但能显著抑制NE、DXM引起的肌管泛素系统基因高表达(p＜0.05或p＜0.01)。结论：在本研究中,小剂量丙泊酚持续镇静具有以下效应：1.改善严重烫伤后的应激紊乱；2.抑制严重烫伤后主要炎症介质的释放,调节促炎／抑炎细胞因子平衡；3.减轻严重烫伤后的脏器损伤,改善肝肾功能；4.抑制严重烧伤、高儿茶酚胺、高皮质醇水平所引起的骨骼肌泛素系统基因的高表达,可能是减轻骨骼肌损伤的主要机制之一。小剂量丙泊酚用于严重烫伤后持续镇静是一种安全有效的治疗方案,不是诱发PRIS危险因素。更多还原

【Abstract】 Objective:Propofol can effectively prevent and regulate dyserethism, excessive inflammatory response, immunologic imbalance, metabolic disturbance and improve the survival rate of critical patients through multiple links after severe burns or trauma. However, in recent years it was reported in some literatures that high-dose (> 4mg·kg-1·h-1) and long-term (> 48h) propofol infusion may induce "propofol infusion syndrome (PRIS)" with low incidence but high mortality, so its safety for sedation of critical patients was challenged. It was found that rhabdomyolysis (RM) played an important role in the occurrence and development of PRIS and the ubiquitin-proteasome pathway played a key role in skeletal muscle damage and dysbolism. This study adopted animal model and in vitro cell culture, and used the low-dose propofol as continuous sedation for burns to observe:1. the impact on releases of stress hormones and inflammatory mediators; 2. the impact on blood gas, blood biochemistry and histopathology of major organ; 3. the impact on the skeletal muscle structure and gene expression of ubiquitin system. This study also systematically evaluated the safety and effectiveness of continuous sedation by low-dose propofol for severe burns, and studied the influence on ubiquitin system of skeletal muscle from the level of gene, providing the theory basis of sedation for severely burned patients using propofol.Material and Methods:1. Experiments on Animals(1) Twenty four healthy oryctolagus cuniculuses were randomly divided into scalding sedation group, scalding control group, sham injury sedation group and sham injury control group. Scalding groups were suffered from 30%TBSA full thickness injury with boiling water after anesthesia, sham injury groups were immerged in warm water (37℃) after anesthesia and then were treated same as scalding groups.3h after scalding/sham injury, sedation groups were constantly infused with 6.2 mg·kg-1·h-1 (2 mg·kg-1·h-1 equivalent of human)1% propofol for 48h, control groups were constantly infused with normal saline of same volume and rate for 48h. Blood samples were taken and preserved before scalding,3h after scalding,6h after scalding (3h after sedation),9h after scalding (6h after sedation) and 51h after scalding (48h after sedation); anesthetized the animals after 48h, the heart, lung, liver, kidney, intestine and skeletal muscle tissue samples and urine sample were preserved. (2) determined concentrations of major stress hormone in blood plasma, inflammatory mediators in blood serum and blood glucose; (3) determined blood gas, blood biochemistry and concentrations of muscle hemoglobin in blood serum and urine; (4) observed the pathological changes of major organ tissue by optical microscope and the ultramicroscopic structure changes of skeletal muscle tissue by transmission electron microscope; (5) determined the gene expression changes of relevant component genes in ubiquitin-proteasome pathway of skeletal muscle tissue.2. In Vitro Cell Experiments(1) Established stable method on separation, culturing, purifying and fusion of satellite cells of rabbit skeletal muscle. (2) Co-cultured the mature myotube with propofol (2μg/ml), dexamethasone (DXM,100μmol/ml) and norepinephrine (NE,10-’mmol/L) for 48h respectively or together, and determined the gene expression changes of relevant component genes in ubiquitin-proteasome pathway of myotube.Results:1. Epnephrine, adrenocorticotropic hormone (ACTH) and insulin in blood plasma were reduced (p<0.05), cortisol was constantly increased (p<0.05) after severe scalding injury; cortisol of scalding sedation group was higher than that of scalding control group after propofol sedation (p< 0.05), and ACTH of sham injury sedation group was reduced compared with sham injury control group (p<0.05) after sedation; 2. TNF-a, IL-1 and IL-10 in blood serum were all increased after severe scalding injury (p<0.05), and propofol sedation may reduce the increasing (p< 0.05); 3. Compared with control group, propofol sedation had no significant influence on PCO2 and PO2 of sedation group(p> 0.05), lactic acid and glucose level in blood of scalding sedation group were decreased compare with scalding control group(p< 0.05); 4. Propofol sedation relieved the increasing of contents of ALT, AST, CK, CK-MB and muscle hemoglobin in blood serum and urine after severe scalding injury (p< 0.05 or p <0.01), and had no significant influence on sham injury group (p> 0.05), but blood cholesterol and triglyceride increased after continuous propofol sedation (p<0.05).5. Heart, lung, liver, kidney, intestine and skeletal muscle were all damaged in varying degrees after severe scalding injury, propofol may attenuate the damage and had no notable influence on sham injury animals; 6. Gene expressions of E2-14KDa enzyme, E3a enzyme and C2 subunit were enhanced in skeletal muscle after severe scalding injury, propofol sedation reduced its high gene expressions effectively (p<0.05 or p<0.01) and had no influence on gene expressions of sham group (p>0.05); 7. Propofol had no influence on gene expression of myotube ubiquitin system when it cultured with myotube alone(p >0.05), but propofol restrained the high gene expression of various component gene of ubiquitin system induced by NE and DXM (p<0.05 or p<0.01).Conclusion:The effects of continuous sedation by low-dose propofol are as following:1. Continuous sedation by low-dose propofol may improve dyserethism after severe scalding injury; may restrain the releasing of main inflammatory factors after severe scalding injury, and regulate the balance of pro-inflammatory factors and anti-inflammatory factors; may attenuate organ injuries after severe scalding injury and improve liver function; may restrain the high gene expressions of skeletal muscle ubiquitin system caused by severe scalding injury and high level of catecholamine and cortisol, and this may be one of its main functions to decrease skeletal muscle injuries.It is a safe and effective treatment to take low-dose propofol for continuous sedation for severe burns, and has no risk to induce PRIS.更多还原