双下肢远程缺血预处理对兔肺缺血再灌注损伤细胞凋亡的影响

发布时间：2018-03-31 06:22

  本文选题：远程缺血预处理　切入点：缺血再灌注损伤　出处：《兰州大学》2010年硕士论文

【摘要】： 目的采用兔单肺原位缺血再灌注损伤模型,通过对双下肢远程缺血预处理后兔单肺原位缺血再灌注损伤引起的肺组织病理学改变、MDA、SOD、细胞凋亡指数(AI) Bcl-2/Bax及血气分析的变化,探讨双下肢远程缺血预处理对兔单肺原位缺血再灌注损伤细胞凋亡的影响及可能机制。 方法清洁级日本大耳白兔18只,体重2.0-2.5kg,随机分为3组(n=6)：假手术对照组(S组)、肺缺血再灌注组(IR组)、肢体远程缺血预处理组(R组)。参照Sekido介绍的方法(略加改造)复制兔肺缺血再灌注模型：于胸骨旁左侧肋软骨与肋骨交界处向头侧剪断第8-6肋骨,开胸暴露左肺门并留置阻断带,在呼气末阻断左肺门60min为缺血期,随后开放阻断带恢复左肺供血和通气为再灌注期。阻断左肺门前经耳缘静脉给予低分子肝素1 mg·kg-1抗凝。远程缺血预处理组(R组)在开胸左肺门放置阻断带稳定30min后用阻断带在兔双下肢根部同时阻断下肢血流造成双下肢缺血10min,以多普勒血流探测仪检测不到股动脉搏动为准,然后松开阻断带再灌10min,重复三次。30min后结扎左肺门60min,然后恢复灌注180min。缺血再灌注组(IR组)在左肺门放置阻断带后旷置90min,在呼气末阻断左肺门60min制造肺缺血再灌注模型为缺血期,以左肺不张为准,60min后松开结扎线,恢复灌注1 80min,只在双下肢松绕阻断带但不行双下肢缺血。假手术组(S组)只在左肺门放置阻断带但不结扎肺门及只在双下肢松绕阻断带但不行双下肢缺血。分别在远程缺血预处理前,再灌注15min、30min、60min、120min及180min时采动脉血测血气并观察有创动脉血压的变化。各组分别于再灌注180min放血处死家兔留取标本待测肺组织MDA含量、SOD活性、HE染色下观察肺损伤定量评价指标(IQA)的变化以及TUNEL法检测肺组织凋亡指数(AI)、Westernbloting法半定量Bcl-2、Bax蛋白的表达。实验过程中监测直肠温度并用电热毯维持直肠温度在36-38℃。 结果①PaO2, A-aDO2和RI:三组的基础A-aDO2、PaO2及RI无统计学差异(P＞0.05),随着再灌注时间的延长,IR组的PO2逐渐降低,尤以再灌注60min内明显(P0.01)。R组和S组的PaO2均高于IR组(P0.01)。IR组的A-aDO2和RI逐渐增加,尤其在再灌注60min时明显高于R组和S组(P0.01)。再灌注后IR组的A-aDO2和RI明显高于R组和S组(P0.01)。②IQA:IR组的IQA明显高于R组和S组(P0.01)。③MDA含量和SOD活性：IR组的MDA含量明显高于S组和R组(P0.01),而IR组的SOD活性明显低于S组和R组(P0.01)。④AI和Bcl-2/Bax:与S组比较,IR组的AI和Bax蛋白含量明显增加(P0.01),但Bcl-2明显降低(P0.01),并且Bcl-2/Bax降低(P0.01)。与IR组比较,R组的AI和Bax明显降低(P0.01),而Bcl-2和Bcl-2/Bax明显增加(P0.01)。R组和S组之间比较无统计学意义(P0.05)。 结论双下肢远程缺血预处理对兔肺缺血再灌注损伤具有保护作用,其保护作用主要是通过抑制氧化损伤,上调Bcl-2/Bax的比值,从而减少肺组织细胞凋亡的发生。
[Abstract]:Objective to study the changes of Bcl-2/Bax and blood gas in lung histopathological changes induced by in situ ischemia-reperfusion injury in rabbits with single lung ischemia reperfusion injury after long leg ischemia preconditioning. To investigate the effect of remote ischemic preconditioning on apoptosis of rabbit single lung ischemia reperfusion injury and its possible mechanism. Methods Eighteen Japanese white rabbits of clean grade were used. Weight 2.0-2.5kg, randomly divided into three groups: sham operation control group S group, lung ischemia reperfusion group IR group, limb remote ischemic preconditioning group R group. According to the method introduced by Sekido (slightly modified), the rabbit lung ischemia-reperfusion model: in chest. Cut the 8-6 ribs at the junction of the left costal cartilage and the ribs at the left side of the bone to the head. Open chest exposed the left pulmonary hilum and retained the blocking band. The left pulmonary hilus 60min was blocked at the end of the breath for ischemic period. The left pulmonary portal vein was blocked with low molecular weight heparin (1 mg kg-1) for anticoagulation.The remote ischemic preconditioning group (group R) placed a blocking zone to stabilize 30min in the open left pulmonary hilum. The femoral artery pulsation could not be detected by Doppler blood flow detector after the occlusion band was used to block the lower extremity blood flow at the same time in both lower extremities of rabbits for 10 min, and the femoral artery pulsation could not be detected by Doppler blood flow detector. The left pulmonary hilum was ligated for 60 minutes after repeated for three times for 30 minutes, and then returned to perfusion for 180 minutes. In the IR group, the left pulmonary hilus was placed in the left pulmonary hilus for 90 minutes. The model of pulmonary ischemia reperfusion was established by occlusion of the left pulmonary hilus 60min at the end of expiratory period, and the model of pulmonary ischemia reperfusion was established at the end of expiratory period. The ligation line was loosened 60 minutes after the left atelectasis. Recovery perfusion for 180 min, only in both lower extremities loose occlusion zone but not lower extremity ischemia. Sham operation group (S group) only placed in the left pulmonary hilus without ligation of the pulmonary hilus, and only in the lower extremities around the blocking zone but not in both lower extremities ischemia. Before remote ischemic preconditioning, The arterial blood gas was measured and the changes of blood pressure of invasive arteries were observed during 15 minutes of reperfusion for 30 minutes and 60 minutes for 120 minutes and for 180min. The quantitative evaluation of lung injury was observed in each group of rabbits who were sacrificed by reperfusion 180min for the determination of MDA content and the activity of MDA and HE staining. The changes of IQA) and the expression of Bax protein in lung tissue were detected by TUNEL. The rectal temperature was monitored and the rectal temperature was maintained at 36-38 鈩，

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