失血性休克诱发兔心肌损伤时缝隙连接蛋白43表达的变化及丙泊酚的干预效应

发布时间：2018-05-06 00:06

本文选题：丙泊酚 + 休克　；参考：《河北医科大学》2016年硕士论文

【摘要】：目的:失血性休克(Hemorrhagic shock,HS)常见于严重创伤和手术出血,是临床上十分危急的病症,若未经及时治疗,可导致死亡。HS主要以全身血容量骤减、组织灌注减少、细胞缺氧、无氧代谢增加及乳酸堆积,氧自由基和代谢产物生成增多为主要特征,造成细胞氧化应激损伤,严重者可导致器官功能障碍,甚至衰竭。尽管心肌细胞对缺血缺氧有较强的耐受力,但严重休克时仍会表现为收缩功能下降、心输出量减少,进而加重其他脏器损伤。丙泊酚是临床常用的静脉麻醉药,具有对抗氧自由基和抑制细胞凋亡的作用。研究表明丙泊酚对失血性休克和缺血-再灌注心肌损伤具有明显的保护作用。缝隙连接蛋白43(Connexin 43,Cx43)是心肌细胞缝隙连接的主要蛋白,Cx43的空间分布、数量及磷酸化异常均可影响缝隙连接电耦联,影响心肌同步收缩功能。研究表明,急性心肌缺血后Cx43会发生重构,主要表现为蛋白表达下降,分布模式以及磷酸化的改变。本研究拟探讨丙泊酚对失血性休克兔心肌损伤时Cx43表达的影响,为明确丙泊酚心肌保护的作用机制提供参考。方法:健康清洁级雄性新西兰白兔24只,5~7月龄,体重2.5~3.0 kg,由河北医科大学实验动物中心提供。采用随机数字表法,将其分为3组(n=8):假手术(S)组、失血性休克(HS)组及丙泊酚处理(P)组。取兔称重后置于兔笼中沿右侧耳缘静脉注射3%戊巴比妥钠30mg/kg,麻醉成功后将兔仰卧位固定于手术台上。手术部位备皮消毒,1%利多卡因浸润麻醉后剪开颈部正中皮肤,分离气管,行气管切开插管术(ID=3.0mm)并保留自主呼吸;分离右侧颈总动脉,用24G套管针插入右侧颈总动脉并用丝线固定,连接M1567A有创血压测压套装,再与Philips G60多功能监护仪连接持续监测动脉血压(BP)和平均动脉压(MAP)。1%利多卡因浸润麻醉后于兔左、右侧股三角区分别斜行3cm剪开皮肤,暴露并分离双侧股动静脉,用24G套管针分别置入左侧股动脉和右侧股静脉并用丝线固定,连接三通用于放血、采集动脉血样和输血、给药。置管完成后静脉给予肝素钠3mg/kg。S组仅行气管切开插管术、右侧颈总动脉和股动静脉穿刺置管术,不放血;P组于放血前10min经右侧股静脉注射丙泊酚5 mg/kg,并用微量注射泵以20mg·kg~(-1)·h~(-1)的速度持续输注至休克后120min;S组及HS组静脉输注等容量生理盐水。采用Wigger’s改良法经左侧股动脉放血,10 min内使MAP降至40mm Hg,放血量约为全身血容量的30~50%,观察兔血压波动,通过放血和回输血液维持MAP在35~45 mm Hg之间以达休克状态并维持120 min,制备失血性休克动物模型。于放血前即刻(T_0)、休克30 min(T_1)、60 min(T_2)、90 min(T_3)和120min(T_4)采集动脉血样测定各时间点血浆cTnI浓度及动脉血乳酸水平。于T_4采血后,连接HX-200动物呼吸机行机械通气,快速打开胸腔,取心尖部心肌组织并一分为二,一半置于EP管中液氮冻存24h,再转-80℃冰箱保存待测;另一半组织用4 4%℃多聚甲醛溶液固定待测。采用ELISA法测定兔血浆cTnI浓度;采用自动血气分析仪检测动脉血乳酸水平;取4%多聚甲醛溶液固定的心肌组织,常规石蜡包埋切片,HE染色,光学显微镜下观察心肌病理损伤并行损伤评分;取-80℃冰箱冻存的心肌组织采用Western blot法检测兔心肌组织Cx43表达。结果:1 与S组比较,HS组与P组兔血浆cTnI浓度升高(P0.05);与HS组比较,P组兔血浆cTnI浓度降低(P0.05);与T_0比较,HS组和P组T_(1-4)时兔血浆cTnI浓度逐渐升高(P0.05)。2 与S组比较,HS组与P组兔动脉血乳酸水平升高(P0.05);与HS组比较,P组兔动脉血乳酸水平降低(P0.05);与T_0比较,HS组和P组T_(1-4)时兔动脉血乳酸水平逐渐升高(P0.05)。3 S组心肌细胞形态正常,肌纤维排列整齐,细胞核均匀一致;HS组心肌细胞肿胀,纤维排列不规则或断裂,间质内有大量炎性细胞浸润,胞核崩解;P组心肌细胞轻度变性,少量心肌纤维断裂,细胞间隙略增宽,病理损伤较HS组减轻。与S组比较,HS组和P组心肌损伤评分升高(P0.05);与HS组比较,P组心肌损伤评分降低(P0.05)。4 与S组比较,HS组和P组心肌Cx43含量减少(P0.05);与HS组比较,P组心肌Cx43含量增加(P0.05)。结论:1 股动脉放血法可成功制备失血性休克动物模型。2 失血性休克可引起心肌损伤并使心肌细胞Cx43表达减少。3 丙泊酚可减轻失血性休克心肌损伤,其机制可能与上调Cx43表达有关。
[Abstract]:Objective: Hemorrhagic shock (HS) is common in severe trauma and surgical bleeding. It is a very critical disease in clinical. If not treated in time, the death.HS can lead to a sudden decrease in systemic blood volume, decreased tissue perfusion, anoxia, anoxic metabolism and lactic acid accumulation, and increased oxygen free radicals and metabolic products. It is characterized by oxidative stress caused by cell oxidative stress, which can cause organ dysfunction and even failure. Although myocardial cells have strong tolerance to ischemia and hypoxia, however, severe shock will still appear to be a decrease in systolic function, a decrease in cardiac output, and further aggravation of other organs. Propofol is a common clinical intravenous anesthetic. The study shows that propofol has a significant protective effect on myocardial damage in hemorrhagic shock and ischemia-reperfusion. Gap connexin 43 (Connexin 43, Cx43) is the main protein in the gap junction of cardiac myocytes. The spatial distribution, quantity and phosphorylation of Cx43 can affect the connexion of gap junction The study shows that Cx43 can be reconstructed after acute myocardial ischemia, mainly manifested in the decrease of protein expression, distribution pattern and the change of phosphorylation. This study intends to explore the effect of propofol on the expression of Cx43 during myocardial injury in rabbits with hemorrhagic shock, and to provide a clear mechanism for the protection of propofol myocardium. Methods: 24 healthy and clean male New Zealand white rabbits, 5~7 months old and weight 2.5~3.0 kg, were provided by the experimental animal center of Hebei Medical University. Using random digital table method, they were divided into 3 groups (n=8): sham operation (S) group, hemorrhagic shock (HS) group and propofol (P) group. After the rabbit was weighed, the rabbit was injected 3% into the right ear edge vein. After the success of pentobarbital sodium 30mg/kg, the rabbit supine position was fixed on the operating table after the anesthesia was successful. The surgical site was prepared by skin preparation and 1% lidocaine, after the infiltration of lidocaine, the median skin of the neck was cut, the trachea was separated, the tracheotomy intubation (ID=3.0mm) was performed and the spontaneous breathing was retained; the right common carotid artery was separated and the right common carotid artery was inserted with the 24G trocar and used silk thread. Fixation, connecting M1567A with a blood pressure pressure measurement kit, and the continuous monitoring of arterial pressure (BP) and the mean arterial pressure (MAP).1% lidocaine infiltration anesthesia with the Philips G60 multi function monitor in the left of the rabbit, the right femoral triangle slanting 3cm to cut off the skin, exposing and separating the bilateral femoral arteriovenous, and placing the left femoral artery with the 24G trocar needle respectively. The femoral vein and the right femoral vein were fixed with silk thread, connecting three to blood letting, collecting arterial blood samples and blood transfusion, giving the medicine. After the catheter was completed, the heparin sodium 3mg/kg.S group was given only tracheotomy intubation, the right common carotid artery and femoral arteriovenous catheterization were performed, and blood was not released; group P was injected 10min via the right femoral vein before releasing the blood, and the use of propofol was 5 mg/kg. The microinjection pump was injected at the speed of 20mg. Kg~ (-1) and h~ (-1) to 120min, 120min, S group and HS group intravenous infusion. Using Wigger 's improvement method, the left femoral artery was bleed to the left femoral artery, and the MAP was reduced to approximately 50% of the whole body blood volume in the 10 min. The blood pressure fluctuation was observed and maintained by blood letting and blood transfusion. MAP between 35~45 mm Hg and 120 min was used to produce hemorrhagic shock animal model. The plasma concentration and arterial blood lactate level at each time point were measured at immediately (T_0), shock 30 min (T_1), 60 min (T_2), 90 min (T_3) and arterial blood samples. Mechanical ventilation, quickly open the chest, take the apex of the myocardial tissue and divide into two, half of the liquid nitrogen in the EP tube in the EP tube to freeze 24h, then turn -80 C refrigerator to be kept for test; the other half of the tissue was fixed at 44% centigrade polycondensation. The ELISA method was used to determine the plasma cTnI concentration in the rabbit; the serum lactate level was detected by the automatic blood gas analyzer; and the concentration of the blood lactate was detected by the automatic blood gas analyzer. Formaldehyde Solution fixed myocardial tissue, conventional paraffin embedded section, HE staining, observation of myocardial pathological injury parallel injury score under optical microscope, and Western blot method was used to detect Cx43 expression of myocardium tissue in the myocardium of freezer at -80 C. Results: 1 compared with group S, the concentration of cTnI in HS group and P group increased (P0.05), and compared with HS group. Compared with the P group, the plasma concentration of cTnI decreased (P0.05). Compared with T_0, HS and P group T_ (1-4) increased the plasma cTnI concentration in rabbits (P0.05).2 and S group, and the level of lactic acid in the arterial blood of the rabbits was higher than that in the S group; compared with the group, the lactate level of the rabbit arterial blood decreased. In the group of.3 S, the myocardial cells were normal, the muscle fibers were arranged and the nuclei were uniform. The myocardial cells in the group HS were swollen, the fiber arrangement was irregular or broken. There were a large number of inflammatory cells infiltrating in the interstitium and the disintegration of the nucleus; the myocardial cells in the P group were slightly denatured, a small amount of myocardial fibers were broken, the gap of the cells increased slightly, and the pathological injury was less than that of the HS group. Compared with group S, myocardial injury scores in group HS and group P were increased (P0.05). Compared with group HS, myocardial injury score of P group decreased (P0.05).4 and S group, HS group and P group decreased. Conclusion: 1 femoral artery bleeding method can be successfully prepared for hemorrhagic shock animal model. Sexual shock can cause myocardial injury and reduce the expression of Cx43 in cardiac myocytes by.3 propofol, which may reduce myocardial injury in hemorrhagic shock, and its mechanism may be related to the up regulation of Cx43 expression.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R614

【参考文献】