全肝缺血再灌注致大鼠肺脏损伤及其防护的实验研究

发布时间：2018-03-20 01:18

  本文选题：肺损伤　切入点：硫化氢　出处：《中国人民解放军军医进修学院》2010年硕士论文　论文类型：学位论文

【摘要】： 背景和目的： 肝脏移植术、肝脏大部切除术临床上极为常见,术中造成全肝缺血再灌注损伤,势必给其它远隔脏器带来损伤,肝缺血及门静脉瘀血可产生大量有害介质,在肝血流恢复后经由心脏立即倾注入肺,造成急性肺损伤,给移植成功带来了巨大的困难,严重影响着术后病人的存活率。 本实验通过手术方法建立大鼠全肝缺血再灌注模型,从术后肺脏结构及功能改变,肺内各类致伤因子的动态变化,以及钙调神经磷酸酶、硫化氢／胱硫醚-γ-裂解酶、水通道蛋白、线粒体能量代谢的改变等方面,较全面研究创伤后肺损伤的发病机制。并应用硫氢化钠、他克莫司、螺内酯、甲泼尼龙进行干预,进一步研究术后肺脏损伤的机制及药物的作用途径,以寻找有效的干预措施。 本实验分为两部分：一、全肝缺血再灌注致大鼠肺脏损伤机制及药物干预对其影响；二、全肝缺血再灌注大鼠肺脏损伤的线粒体机制及其药物干预 方法：通过手术方法建立大鼠全肝缺血再灌注的创伤模型。 第一部分：雄性Wistar大鼠184只,随机分为23组,每组8只：正常对照组、缺血20min再灌注0h、2h、6h、12h、24h、48h、72h组、缺血40分钟再灌注0h、6h、72h组；FK-506、MP、NaHS、Spiron空白对照组及20min+6h干预组;MP、Spiron 40min+6h及40min+72h干预组。第二部分：雄性Wistar大鼠80只,随机分为10组,每组8只：正常对照组、缺血20min再灌注6h组、FK-506、MP、NaHS、Spiron空白对照组及20min+6h干预组。 动态检测各组大鼠血浆硫化氢(H2S)、丙二醛(MDA)、血管紧张素II(AngⅡ)、醛固酮(ALD)、肿瘤坏死因子-a(TNF-a)、白介素-8(IL-8)的变化,检测肺脏湿干重比值、CSE活性,细胞间粘附分子(ICAM-1)、核因子NF-κB、IL-8、AQP1含量,钙调神经磷酸酶(CaN)含量、活性及mRNA表达,测定线粒体呼吸功能、膜电位及粒体酶的变化,观察光镜及电镜下肺组织形态结构的变化。 结果： 第一部分：与正常对照组比较,缺血20min再灌注组,血清TNF-a、血清及肺脏IL-8、血浆MDA、血浆AngⅡ、血浆H2S、肺脏W/D、ICAM-1、NF-κB水平在缺血期即开始升高,再灌注后持续升高,于再灌注2-12h之间各达到峰值后降低；肺脏AQP1蛋白表达缺血再灌注后逐渐降低,于再灌注6h时降到最低值；肺脏CSE、血浆及肺脏ALD、肺脏CaN含量、活性及mRNA表达缺血再灌注后升高,分别于2h、6-12h出现两次峰值后逐渐下降,多数监测指标于再灌注后72h恢复至接近正常值水平；光镜及电镜结果显示缺血再灌注后6h肺脏组织损伤最为严重。缺血40min再灌注组表现相同的变化趋势,大多数指标变化程度较缺血20min相应组更为显著,肺组织损伤较缺血20min组更为严重；应用FK506、Spiron、MP、NaHS药物干预后,观察到创伤最重时间点的肺组织各项指标出现不同程度改善,提示药物对肺组织损伤起到了不同程度的保护作用。第二部分：与正常对照组比较,缺血再灌注后,肺脏线粒体ATP酶活性改变,RCR、P/O、膜电位均降低,应用FK506、Spiron、MP、NaHS药物干预后,各项指标均有不同程度的改善。 结论： 全肝缺血再灌注模型中,缺血和再灌注两个阶段均可造成肺脏损伤,且随缺血时间延长,肺组织损伤加重,肺脏损伤以再灌注6h损伤最为严重。各种致伤因子作用于肺组织引起损伤,损伤过程中,钙调神经磷酸酶、内源性H2S/CSE这两条信号转导通路的改变,水通道蛋白的减低,线粒体能量代谢障碍均参与了肺脏的损伤,是创伤致肺损伤的重要病理生理学基础之一。应用FK506、Spiron、MP、NaHS药物干预后,药物通过作用于上述机制对缺血再灌注所致肺损伤起到不同程度的保护作用。
[Abstract]:Background and purpose:
Liver transplantation, liver resection on clinical is extremely common, resulting in hepatic ischemia reperfusion injury, is bound to the other remote organ damage, liver ischemia and portal vein blood stasis can produce a lot of harmful medium in the liver after restoration of blood flow through the heart immediately poured into the lungs, causing acute lung injury, to transplant success has brought great difficulties, seriously affected the survival rate of patients after operation.
This experiment established through operation. The total hepatic ischemia-reperfusion in rats model, from the change of lung structure and function after operation, the dynamic changes of all kinds of lung injury factor, and calcineurin / H2S, cystathionine gamma lyase, water channel protein, mitochondrial energy metabolism changes, compared with a comprehensive study of the pathogenesis of lung injury after trauma. And the application of sodium hydrosulfide, tacrolimus, spironolactone, methylprednisolone intervention effect, further study and drug mechanism of lung injury after operation, in order to find out the effective intervention measures.
The experiment is divided into two parts: first, the mechanism of lung injury induced by total hepatic ischemia reperfusion and the effect of drug intervention; two, mitochondria mechanism and drug intervention of lung injury in rats after total hepatic ischemia-reperfusion.
Methods: the traumatic model of rat liver ischemia-reperfusion was established by operation.
The first part: 184 male Wistar rats were randomly divided into 23 groups, 8 rats in each group: normal control group, ischemia reperfusion 20min, 0h, 2h, 6h, 12h, 24h, 48h, 72h group, 40 min of ischemia reperfusion 0h, 6h, FK-506, MP, 72h; NaHS, Spiron control group and 20min+6h intervention group; MP, Spiron 40min+6h and 40min+72h group. The second part: 80 male Wistar rats were randomly divided into 10 groups, 8 rats in each group: normal control group, ischemia reperfusion group, 20min 6h, FK-506, MP, NaHS, Spiron control group and 20min+6h group.
Rat plasma hydrogen sulfide (H2S), dynamic detect malondialdehyde (MDA), angiotensin II (Ang II), aldosterone (ALD), tumor necrosis factor -a (TNF-a), interleukin -8 (IL-8) change detection, lung wet to dry weight ratio, CSE activity, cell adhesion molecule (ICAM-1) nuclear factor kappa B, NF-, IL-8, AQP1 content, calcineurin (CaN) content, activity and expression of mRNA, mitochondrial respiratory function, changes of membrane potential and mitochondrial enzyme, observe the morphological changes in lung tissue under the light microscope and electron microscope.
Result锛，

本文编号：1636864