胸段硬膜外阻滞对大鼠心肌缺血再灌注损伤的保护作用

发布时间：2018-05-02 09:54

本文选题：胸段硬膜外阻滞 + 缺血再灌注损伤　；参考：《新乡医学院》2017年硕士论文

【摘要】：目的通过对雄性Wistar大鼠心肌缺血再灌注损伤前给予胸段硬膜外阻滞处理,采集大鼠心率、心电图及平均动脉压,测定大鼠各时间段之间血清心肌肌钙蛋白I(cTnI)含量,检测心肌组织自噬相关蛋白(Beclin-1)表达水平及TTC染色计算心肌梗死面积,来探讨胸段硬膜外阻滞对大鼠心肌缺血再灌注损伤(MIRI)的保护作用。方法1、研究分组将30只健康成年雄性Wistar大鼠随机分为假手术组、心肌缺血再灌注组(I/R)组和胸段硬膜外阻滞组(TEB)组,每组10只。TEB组、I/R组大鼠结扎左冠状动脉前降支前分别向硬膜外腔给予体积分数0.5%罗哌卡因0.125ml·kg-1和等量生理盐水,假手术组大鼠给予心脏左冠状动脉前降支穿线,但不结扎,穿线前硬膜外腔给予生理盐水0.125ml·kg-1;TEB组和I/R组大鼠给予心脏左冠状动脉前降支穿线并结扎,40min后解开结扎线复灌120min。TEB组大鼠胸段硬膜外阻滞后根据大鼠胸腹肌肉松弛,阻滞区体表温度升高,大鼠左前降支结扎、复灌后,根据心肌大体颜色观察及心电图等提示动物模型制备成功。2、心电图及血流动力学检测:利用多功能生物采集系统,在大鼠体表连接肢体导联,持续检测记录各组大鼠开胸前(T_0)、左冠状动脉结扎前(T_1)、缺血20min(T_2)、缺血40min(T_3)、再灌注120min(T_4)的心率(HR)、平均动脉压(MAP)。3、ELISA法测定血清肌钙蛋白I(cTnI)含量:每只大鼠于开胸前(T_0)、左冠状动脉结扎前(T_1)、缺血20min(T_2)、缺血40min(T_3)及再灌注120min(T_4)时取静脉血,用ELISA法测血清cTnI水平。4、TTC染色:再灌注120min时处死大鼠,取心脏,用冰盐水冲洗干净积血,将心脏放入-20℃冰箱冷冻20min,切成2mm厚的心肌切片,用TTC染色,用Image—Pro Plus6.0图象分析软件(美国Media Cybernetics公司)计算梗死区和心肌缺血危险区面积。5、Western blot方法检测心肌组织自噬相关蛋白(Beclin-1)表达水平:再灌注120min后取心脏,将缺血危险区心肌组织放液氮冷冻后,提取组织蛋白,检测心肌组织自噬相关蛋白(Beclin-1)表达水平。6、统计分析数据:采用SPSS21.0统计软件进行分析,计量资料以均数±标准差((?)±s)表示,行单因素AVONA方差分析,两均数比较采用t检验P0.05为差异有统计学意义。结果1、与组内T_0时及假手术组同时间点比较,T_2~T_4时I/R组、TEB组大鼠HR、MAP显著降低(P0.05);与I/R组同时间点比较,T_2~T_4时TEB组HR、MAP显著升高(P0.05)。与T_0时比较T_1时三组HR、MAP差异无统计学意义(P0.05)。2、与T_0时比较,T_2~T_4时I/R组、TEB组血清cTnI水平显著升高(P0.01);与假手术组同时间点比较,T_2~T_4时I/R、TEB组血清cTnI水平显著升高(P0.01);与TEB组同时间点比较,T_2~T_3时I/R组血清cTnI水平差异无统计学意义(P0.05),T_4时I/R组血清cTnI水平较TEB组显著升高(P0.01),T_0时三组之间比较血清cTnI水平差异无统计学意义(P0.05)。3、与I/R组比较,TEB组心肌梗死区与心肌缺血危险区的百分比显著降低(P0.01),假手术组大鼠无心肌梗死。4、与假手术组比较I/R组大鼠心肌组织中Beclin-1表达显著增高(P0.01),与I/R组比较TEB组大鼠心肌组织中Beclin-1水平显著降低(P0.01)。结论TEB能够减轻MIRI,对心肌有明显的保护作用,其机制可能与下调心肌细胞自噬因子表达,从而减少再灌注阶段心肌细胞自噬有关。
[Abstract]:Objective to measure the heart rate, electrocardiogram and mean arterial pressure of rats before myocardial ischemia reperfusion injury in male Wistar rats, and to determine the content of cardiac troponin I (cTnI) in serum, the expression of autophagy related protein (Beclin-1) and TTC staining for myocardial infarction. The protective effect of thoracic epidural block on myocardial ischemia reperfusion injury (MIRI) in rats was investigated. Methods 1, 30 healthy adult male Wistar rats were randomly divided into sham operation group, myocardial ischemia reperfusion group (I/R) group and thoracic epidural block group (TEB) group, 10.TEB groups in each group, and I/R group ligation of left coronary artery. The volume fraction of 0.5% ropivacaine 0.125ml kg-1 and equal amount of normal saline were given to the epidural cavity before the anterior descending branch of the pulse. The rats in the sham operation group were given the left anterior descending branch of the coronary artery, but they were not ligation and 0.125ml. Kg-1 was given to the epidural cavity before the thread was worn, and the left anterior descending branch of the coronary artery in the group of TEB and the group I/R was given the line of the left anterior descending branch of the coronary artery. After ligating and ligation, after 40min, the thoracic and abdominal muscle relaxation of rats in group 120min.TEB was unraveled according to the relaxation of the thoracic and abdominal muscles of the rats, the body surface temperature in the block was increased, the left anterior descending branch of the rat was ligated. After the reperfusion, the animal model was prepared according to the gross color observation of the myocardium and electrocardiogram, and the electrocardiogram and hemodynamic tests were made: using the multifunction test. The biological collection system was used to connect the limb lead on the body surface of the rat. The rats were continuously recorded before the chest (T_0), the left coronary artery ligation (T_1), ischemic 20min (T_2), ischemic 40min (T_3), and reperfusion 120min (T_4) heart rate (HR), mean arterial pressure (MAP).3, and ELISA method to determine the content of serum troponin. Before ligation of the left coronary artery (T_1), ischemic 20min (T_2), ischemic 40min (T_3) and reperfusion 120min (T_4), the venous blood was taken. The serum cTnI level.4, TTC staining was measured by ELISA method. Image - Pro Plus6.0 image analysis software (American Media Cybernetics company) was used to calculate the area.5 in the infarct area and the area of myocardial ischemia. The Western blot method was used to detect the expression level of autophagy related protein (Beclin-1) in myocardial tissue: after reperfusion 120min, the heart was taken, and the tissue protein was extracted after the cryosurgery of the myocardial tissue of the ischemic risk area and detected the tissue protein. Myocardial autophagy related protein (Beclin-1) expression level.6, statistical analysis data: using SPSS21.0 statistical software for analysis, measurement data with mean + standard deviation (?) + s), single factor AVONA variance analysis, two average number comparison using t test P0.05 for difference is statistically significant. Results 1, with the same time in the group T_0 and the sham operation group in the same time Comparison, T_2~T_4 I/R group, group TEB rats HR, MAP significantly decreased (P0.05); compared with the I/R group at the same time point, T_2~T_4 TEB group HR, MAP significantly increased (P0.05). The level of serum cTnI in group TEB was significantly higher in I/R and TEB (P0.01), compared with group TEB at the same time, and there was no significant difference in serum cTnI level in I/R group at T_2~T_3 (P0.05). There was no significant difference in serum levels between the three groups at T_4 (P0.05). Compared with the group TEB, the percentage of myocardial infarction area and myocardial ischemia was significantly lower (P0.01). There was no myocardial infarction in the sham operation group (.4). Compared with the sham group, the Beclin-1 expression in the myocardial tissue of the I/R group was significantly higher (P0.01), and the Beclin-1 level in the myocardial tissue of the TEB group was significantly lower than that of the I/R group (P0.01). Conclusion TEB could be reduced. Light MIRI has an obvious protective effect on myocardium, and its mechanism may be related to downregulation of autophagy factor expression in myocardium, thereby reducing autophagy in myocardial cells at reperfusion stage.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

【参考文献】