血管内皮生长因子对犬体外循环肺损伤的保护作用及机制研究

发布时间：2018-01-11 01:03

本文关键词：血管内皮生长因子对犬体外循环肺损伤的保护作用及机制研究　出处：《西南医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景及目的:随着我国社会经济的变革和人们生活方式的变化及人口趋于老龄化,心血管病发病的危险因素持续增长,其死亡率占总死亡原因的首位。体外循环(cardiopulmonary bypass,CPB)心脏直视手术是目前治疗心脏器质性病变的主要手段之一,尽管体外循环技术在设备、材料和心外科手术技术等方面已经取得了巨大进步,体外循环下心内直视手术的并发症和病死率逐步下降,但由于其本身的非生理性的血流动力学特点以及低温、低灌注、炎症反应等所造成各个器官和系统的损伤仍是体外循环术后常见的并发症之一。凡是经历CPB的患者术后均有不同程度的肺功能减退,轻者仅有一过性症状,重者可出现呼吸窘迫甚至急性呼吸衰竭,造成不可挽救的后果。因此,如何避免体外循环中的肺损伤一直是人们关注的重点。血管内皮损伤是各种缺血再灌注和炎性反应导致器官损伤的重要途径,作为血管修复因子的血管内皮生长因子(vascular endothelial growth factor,VEGF)目前被广泛用于相关的研究。目前已有研究证明体外循环时使用血管内皮生长因子(VEGF)可以对缺血再灌注的心脏、肾脏有一定的保护作用,但VEGF对体外循环后肺损伤是否有保护作用研究较少。本研究设想利用外源性的VEGF干预来减轻CPB后肺损伤,故通过建立犬体外循环模型,观察外源性VEGF对CPB后肺损伤的保护作用,旨在为CPB后肺保护提供新方法。方法:选择12-15kg的雄性Beagle犬9只,术前12h禁食水,阿托品0.5mg术前30min肌肉注射,异戊巴比妥钠3-5mg/kg、丙泊酚3mg/kg静脉注射麻醉后迅速用6.5#导管行气管插管,连接至呼吸机(吸入氧浓度100%,潮气量15ml/kg,呼吸频率17次/分)。股动脉穿刺后连接心电监护仪,用7F中心静脉导管置入颈内静脉,用于监测静脉压和泵入血管活性药物。常规建立体外循环。体外循环转流时间120min,复跳后辅助循环30min。保持转机温度32~34℃,维持平均体循环压60-70mm Hg,体外循环流量80~120ml/(kg·min),通过自体输血维持血细胞比容(Hematocrit,Hct)于24-28%之间。所有动物随机分成假手术组(sham组)、对照组(con组)和实验组(vegf组),每组各3只。sham组在手术前30min内缓慢静脉泵入生理盐水0.5g/kg,再予以开胸及主动脉、上下腔静脉插管,不行体外循环转流。con组在手术前30min内缓慢静脉泵入生理盐水0.5g/kg,常规体外循环转流120min,辅助循环30min。vegf组手术前30min内缓慢静脉泵注vegf 0.5g/kg,常规体外循环转流120min,辅助循环30min。实验结束后取相同部位肺组织计算肺湿/干重(W/D)比值;并取一定量肺组织制成肺组织匀浆,离心后取上清液,分别测定MDA、SOD、IL-6及TNF-α含量。取一定量的肺组织固定、切片、HE染色后光镜下观察病理学结果。结果:1、W/D比值:体外循环后con组的W/D值(11.3±1.6)明显高于sham组(6.3±0.8),P=0.003,差异具有统计学意义;con组的W/D值明显高于vegf组(7.8±1.2),P=0.014,差异具有统计学意义;vegf组的W/D值稍高于con组,P=0.114,差异不具有统计学意义。2、MDA含量:体外循环后con组的MDA含量(268.121±4.424 ng/ml)较sham组(227.336±3.948 ng/ml)有明显升高,P=0.001,差异具有统计学意义;vegf组的MDA含量(246.488±8.585ng/ml)明显高于sham组,P=0.021,差异具有统计学意义;而con组的MDA含量明显高于vegf组,P=0.009,差异具有统计学意义。3、SOD含量:sham组的SOD含量(1622.126±36.212 ng/ml)明显高于con组(1364.391±92.761 ng/ml),P=0.006,差异具有统计学意义;vegf组的SOD含量(1541.142±25.539 ng/ml)明显高于con组,P=0.018,差异具有统计学意义;sham组的SOD含量稍高于vegf组,P=0.218,差异不具有统计学意义。4、IL-6水平:体外循环后con组的IL-6水平(135.851±10.276 ng/ml)明显高于sham组(106.129±1.986 ng/ml),P=0.005,差异具有统计学意义;con组的IL-6水平明显高于vegf组(114.915±3.561 ng/ml),P=0.014,差异具有统计学意义;vegf组的IL-6水平稍高于sham组,P=0.224,差异不具有统计学意义。5、TNF-α含量:体外循环后con组的TNF-α含量(247.675±1.735 ng/ml)较sham组(206.798±2.414 ng/ml)有明显升高,P=0.001,差异具有统计学意义;vegf组的TNF-α含量(222.618±9.000 ng/ml)较sham组有明显升高,P=0.032,差异具有统计学意义;con组的TNF-α含量明显高于vegf组,P=0.003,差异具有统计学意义。6、肺组织病理学:con组肺组织水肿、充血,肺泡间隔增宽,大量炎性细胞和红细胞浸润明显,vegf组肺组织轻度水肿,少量炎细胞浸润,而sham组的肺组织病理学改变明显轻于con组及vegf组。结论:1、本实验利用犬建立体外循环模型,操作简单,有效的模拟了人体体外循环后的病理生理状况,实验重复性好,稳定度高。2、血管内皮生长因子(VEGF)可以降低犬体外循环期间因肺缺血再灌注引起的肺微血管通透性增加,减少膜脂质过氧化反应,增加机体清除自由基的能力。3、血管内皮生长因子对体外循环后肺损伤有一定的保护作用,可能系因其可修复血管内皮损伤,减少肺部缺血缺氧状况,减少氧自由基的破坏和抑制炎性细胞因子的聚集和激活,从而达到肺保护的目的。
[Abstract]:Background and objective: with the change of population and the change of our social economy and people's life style tends to be aging, the risk factors of cardiovascular disease continues to grow, the mortality rate of the total causes of death first. CPB (cardiopulmonary bypass, CPB) in open heart surgery is the major treatment of cardiac disorders at present although, extracorporeal circulation technology in equipment, materials and cardiac surgery technology and other aspects have made great progress, cardiopulmonary bypass heart surgery complications and mortality rate gradually declined, but because the hemodynamic characteristics of non physiological itself and the low temperature, low perfusion injury is a common complication of cardiopulmonary bypass in all the organs and systems caused by inflammation. All CPB patients experience postoperative pulmonary function showed different degrees of decline, only a few light There may be severe respiratory distress symptoms, acute respiratory failure and even cause irreparable consequences. Therefore, how to avoid the lung injury during cardiopulmonary bypass has been the focus of attention. Vascular endothelial injury of ischemia reperfusion and various inflammatory reaction leads to an important way of organ damage, as vascular repair factor, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is currently widely used in related research. At present it has been shown that the bypass using vascular endothelial growth factor (VEGF) on ischemia reperfusion injury of heart and kidney has certain protective effect, but VEGF on pulmonary injury after cardiopulmonary bypass have protective effect on this research is less. Envision the use of exogenous VEGF intervention to reduce lung injury after CPB, so through the establishment of cardiopulmonary bypass model, the effect of exogenous VEGF on lung injury after CPB And to provide new methods for lung protection after CPB. Methods: 12-15kg male Beagle dogs 9, preoperative fasting 12h, atropine 0.5mg 30min preoperative intramuscular injection of amobarbital sodium 3-5mg/kg, propofol 3mg/kg anesthesia rapidly after intravenous injection with 6.5# catheter connected to the tracheal intubation, ventilator (inhaled oxygen concentration 100% 15ml/kg, tidal volume, respiratory rate 17 / min). After puncture of femoral artery connecting ECG monitor, 7F central venous catheter into internal jugular vein for venous pressure monitoring, and infusion of vasoactive drugs. Routine extracorporeal circulation. The CPB time 120min, 30min. transfer assisted circulation temperature 32~34 Rebeating, maintain average the systemic circulatory pressure 60-70mm Hg, extracorporeal circulation flow 80~120ml/ (kg - min), maintaining hematocrit by autologous blood transfusion (Hematocrit, Hct) in 24-28%. All animal random divided into sham operation group (Group sham), The control group (CON group) and experimental group (VEGF group), each group had 3 rats in group.Sham before operation 30min slow intravenous infusion of normal saline 0.5g/kg, and then be open chest and aorta, inferior vena cava intubation, no cardiopulmonary bypass group.Con before the operation in 30min slow intravenous infusion of saline 0.5g/kg the conventional cardiopulmonary bypass, 120min, 30min.vegf group before surgery assisted circulation 30min slow intravenous infusion of VEGF 0.5g/kg, conventional cardiopulmonary bypass 120min, the end of 30min. assisted circulation after the experiment from the lung tissue the same parts of the lung wet / dry weight ratio (W/D); and taking a certain amount of lung tissue into lung homogenate and centrifugation after the supernatant were measured, MDA, SOD, IL-6 and TNF- alpha content. Take a certain amount of lung tissue fixation, slice, HE staining was observed under pathological results. Results: 1, the ratio of W/D: con after cardiopulmonary bypass group W/D (11.3 + 1.6) was significantly higher than that of sham group (6 .3 + 0.8, P=0.003), the difference was statistically significant; the W/D value of con group was significantly higher than that of VEGF group (7.8 + 1.2), P=0.014, the difference was statistically significant; the W/D value of VEGF group was slightly higher than that of group con, P=0.114, the difference was not statistically significant.2, MDA content: the content of MDA in group con after CPB (268.121 + 4.424 ng/ml) than in sham group (227.336 + 3.948 ng/ml) significantly increased, P=0.001, the difference was statistically significant; the content of MDA in group VEGF (246.488 + 8.585ng/ml) was significantly higher than that of group sham, P=0.021, the difference was statistically significant; while the content of MDA in con group was significantly higher than that of group VEGF, P=0.009, the difference was statistically the significance of.3, SOD content: the content of SOD in group sham (1622.126 + 36.212 ng/ml) was significantly higher than that of group con (1364.391 + 92.761 ng/ml), P=0.006, the difference was statistically significant; the content of SOD in group VEGF (1541.142 + 25.539 ng/ml) was significantly higher than that of group con, P=0.018, the difference has statistical meaning Meaning; SOD content of group sham was slightly higher than that of group VEGF, P=0.218, the difference was not statistically significant.4, IL-6 level: the level of IL-6 in group con after CPB (135.851 + 10.276 ng/ml) was significantly higher than that of group sham (106.129 + 1.986 ng/ml), P=0.005, the difference was statistically significant; the level of IL-6 in con group was significantly higher than that of VEGF group (114.915 + 3.561 ng/ml), P=0.014, the difference was statistically significant; the IL-6 level of VEGF group was slightly higher than that of group sham, P=0.224, the difference was not statistically significant.5, TNF- alpha content: after CPB in group con TNF- were (247.675 + 1.735 ng/ml) than in sham group (206.798 + 2.414 ng/ml) significantly increased P=0.001, the difference was statistically significant; the TNF- alpha in VEGF group (222.618 + 9 ng/ml) was significantly higher than that in group sham, P=0.032, the difference was statistically significant; the TNF- alpha con group were significantly higher than that in group VEGF, P=0.003, the difference was statistically significant.6, Lung Group Tissue pathology: con group lung tissue edema, congestion, alveolar septum, inflammatory cells and red blood cell infiltration, lung tissue in VEGF group was mild edema, infiltration of inflammatory cells, and pulmonary pathological changes in sham group was lower than that in the con group and VEGF group. Conclusion: 1. This experiment the establishment of extracorporeal circulation model, the dog has the advantages of simple operation, effective simulation of the pathophysiology of human body after cardiopulmonary bypass, good repeatability, high stability of.2, vascular endothelial growth factor (VEGF) can reduce the increase during cardiopulmonary bypass in dogs caused by pulmonary ischemia reperfusion injury of pulmonary microvascular permeability, reduce membrane lipid peroxidation. Increase the body's free radical scavenging ability of.3, vascular endothelial growth factor has a protective effect on lung injury after cardiopulmonary bypass, probably because of the repair of vascular endothelial injury, reduce lung ischemia and hypoxia condition, reduce oxygen free radical damage And inhibit the accumulation and activation of inflammatory cytokines, so as to achieve the purpose of lung protection.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R654.1;R563

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