心脏缺血预适应对中性粒细胞功能与活性氧释放的影响

Effect of cardiac ischemic preconditioning on the function of neutrophil and release of reactive oxygen

目的:观察缺血预适应中中性粒细胞黏附、浸润及活性氧释放的特点,旨在探讨预适应保护效应与白细胞功能及活性氧的关系。方法:①实验于2000-06/2001-12在解放军广州军区广州总医院医学实验科及南方医科大学南方医院心内科实验室完成。选用杂种犬12条,雄性7条,雌性5条。②麻醉后,开胸游离前降支冠状动脉,稳定30min,造成心肌缺血模型。造模后将犬随机分为2组:缺血再灌注组(n=6),缺血1h后再灌注2h;缺血预适应组(6条),在缺血1h前给予血流阻断5min,灌注5min,反复4次,然后缺血1h,再灌注2h。③分别于基础(缺血和再灌注之前)、缺血1h、再灌注2h各时间点,采用流式细胞术测定中性粒细胞CD11b/CD18表达,按梗死面积%=梗死区面积/危险区面积×100%公式计算心肌梗死范围。按南京聚力生物医学工程提供试剂盒说明测定血清丙二醛水平和超氧化物歧化酶活性。计算髓过氧化物酶活力(kU/g)=ΔA460/(12×组织酶量/3),ΔA460为460nm处吸光度1min增大的值,3为反应的总体积,12为Sigma公司规定的每毫升1mg邻联茴香胺的吸光度;组织酶量指每0.1mL所用酶液中含酶的量。④两组间比较用配对t检验,多组间比较用方差分析,多组间两两比较用SNK方法。结果:犬12条均进入结果分析。①心肌梗死范围:缺血预适应组明显小于缺血再灌注组(P<0.01)。②中性粒细胞膜CD11/CD18表达:缺血再灌注组缺血1h、再灌注2h明显高于基础和缺血预适应组(P<0.05～0.01)。③髓过氧化物酶活性:缺血预适应组缺血和坏死心肌明显低于缺血再灌注组(P<0.05),两组缺血和坏死心肌明显高于正常心肌(P<0.05)。缺血预适应组正常心肌组织低于缺血再灌注组,但差异不明显(P=0.11)。血清丙二醛水平:缺血再灌注组缺血1h和再灌注2h明显高于基础和缺血预适应组(P<0.05)。血清超氧化物岐化酶活性:缺血1h缺血再灌注组明显低于基础和缺血预适应组(P<0.05)。再灌注2h虽然也有此变化,但差异不明显(P>0.05)。结论:缺血预适应能阻抑缺血再灌注所致的中性粒细胞黏附、浸润及活性氧的损害,这可能是发挥缺血预适应保护的重要作用之一。

AIM： To observe the characteristics of adhesion and infiltration of neutrophil and release of reactive oxygen in myocardial ischemic preconditioning and explore the relationship of protective effects of preconditioning with neutrophil function and reactive oxygen. METHODS：（1） The experiment was finished in Department of Experiment, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA and laboratory of Department of Cardiology, Nanfang Hospital, Southern Medical University from June 2000 to December 2001. Twelve hybrid dogs were selected in our investigation, including 7 males and 5 females. （2） The anesthetized open-chest dogs with left descending coronary artery separated were set up then made it steady for 30 minutes to make models with myocardial ischemia, and then they were divided into two groups randomly： Those in the ischemia-reperfusion group （n =6） were subjected to 1-hour occlusion as well as 2 hours reperfusion, while those in the ischemic preconditioning group （n=6） were subjected to 5 minutes occlusion and 5 minutes reperfusion for 4 times before 1 hour occlusion and 2 hours reperfusion. （3） Blood specimens were collected from coronary venous sinus at interval of base （before ischemia and reperfusion）, 1 hour occlusion, 2 hours reperfusion. The expression of neutrophil CD11b/CD18 was measured by flow cytometry. Myocardial infarct extent was calculated according to the formula： percents of infarct area=（infarct area/risk area）×l00%. Level of malondialdehyde （MDA） and activity of superoxide dismutase （SOD） were determined on the basis of the reagent box provided by Nanjing Juli Biologic Medical Engineering Company. Activity of myeloperoxidase （kU/g）= ΔA460（ 12×content of tissue enzyme÷3） ,ΔA460： the value of 1 minute absorbance （A） in 460 nm; 3： the total volume of reaction; 12： the A of 1 mg O-dianisidine dihydrochluride per mililiter provided by Sigma company; content of tisue enzyme： the quantity of myocardial myeloperoxidase contained in per 0.1 mL enzyme liquid. （4） Paired t-test was token to compare between the two groups. Analysis of variance was used in multiple groups. Pairwise comparison in multiple groups was conducted with SNK method. RESULTS： Totally 12 dogs were involved in the result analysis. （1） Myocardial infarct size： It was lower in the ischemic preconditioning group significantly than that in the ischemia-reperfusion group （P 〈 0.01）. （2） The expression of neutrophil CD11b/CD18： It was higher in the ischemia- reperfusion group at both times of 1 hour and 2 hours significantly than at base and in the ischemic preconditioning group （P 〈 0.05-0.01）. （3） Activity of myeloperoxidase： Compared with the ischemia-reperfusion group, it was lower significantly both in ischemic and necrotic myocardium in the ischemic preconditioning group（P 〈 0.05）. It was higher in ischemic and necrotic myocardium than that in normal myoeardium in both groups （P 〈 0.05）. It was lower in normal myocardium than ischemic one in the ischemic-preconditioning group, but the difference was insignificance （P =0.11）. Malondialdehyde level in serum： Compared with base and ischemic preconditioning group, it went up significantly at the time of 1 hour occlusion and 2 hour reperfusion in the ischemia-reperfusion group （P〈 0.05）. Activity of superoxide dismutase： Compared with base and ischemic preconditioning group, it decreased significantly at the time of 1 hour occlusion （P 〈 0.05） and no difference at 2 hour reperfusion in the ischemia-reperfusion group （P 〉 0.05）. CONCLUSION： Myocardial ischemic preconditioning can obviously inhibit the adhesion molecule expression and infiltration of neutrophil as well as the injury of reactive oxygen, which caused by ischemia and reperfusion. These may be composed of one of mechanisms to produce the protection of ischemic preconditioning.