【作者】 程翅；

【导师】 喻田；

【作者基本信息】 遵义医学院 ， 麻醉， 2009， 硕士

【摘要】 目的:采用成年大鼠心肌细胞缺氧复氧模型以及Langendorff离体灌注大鼠心肌缺血再灌注损伤模型,研究硫化氢(以NaHS为供体)后处理对缺血再灌注损伤大鼠心功能与结构的影响,以探索其心肌保护作用的可能机制。方法:1.采用Langendorff装置建立大鼠离体心肌缺血再灌注损伤模型,将SD大鼠随机分为正常组(NOR)、缺血组(I/R)、缺血后处理组(Pcon)、硫化氢后处理组(NaHS1μM、10μM、100μM)、5-羟葵酸拮抗组(5HD)、HMR1098拮抗组(HMR1098),每组8例。正常组:平衡灌注20分钟后续灌100min;缺血组:平衡灌注20分钟,灌注4℃ST.Thomas停跳液,全心缺血40min,复灌60min;缺血后处理组:离体心脏平衡灌注20分钟,全心缺血40分钟,再灌注开始给予6次间隔有氧灌注10秒全心缺血10秒,续灌60分钟;硫化氢后处理组:离体心脏平衡灌注20分钟,全心缺血40分钟,再灌注60分钟,再灌注前给予含各浓度NaHS的K-H液2分钟;5-羟葵酸拮抗组:用药前给予含5-羟葵酸的K-H液5min,余同NaHS 10μM;HMR1098拮抗组:用药前给予含HMR1098的K-H液5min,余同NaHS 10μM。对比观察(1)平衡末、再灌注15分钟、30分钟、45分钟以及灌注末各时点的心功能指标:心率(HR)、左室舒张末压(LVEDP)、左室发展压(LVDP)、压力瞬时最大变化率(dp/dtmax);(2)平衡末、再灌注末取心肌组织并分离、提取线粒体,氧电极分析各组心肌组织线粒体呼吸功能的变化。2.采用Langendorff装置分离成年大鼠心肌细胞,将同批细胞随机分为正常组(NOR)、缺氧组(H/R)、硫化氢后处理组(NaHS 10μM)、缺氧后处理组(IPTC)、5HD拮抗组(5HD)、HMR1098拮抗组(HMR1098)。各组细胞复氧末在激光共聚焦显微镜下对比观察(1)线粒体膜电位的变化;(2)F-肌动蛋白的变化。结果:1.心功能指标的变化:A.平衡末,心功能各项指标各组差异无统计学意义(P＞0.05);B.与平衡末比较,再灌注后各组心功能均呈下降趋势,以I/R组以及5HD组下降较为显著(P＜0.05),而该两组间无明显差异;C.再灌注后NaHS 1μM、NaHS 10μM、NaHS 100μM组与Pcon组比较,各组间心功能指标无明显差异(P＞0.05);D.应用阻断剂的两组,以HMR1098组心功能各项指标恢复较为迅速,与NaHS 10μM比较,其差异无统计学意义(P＞0.05),但明显优于5HD组和I/R组(P＜0.05)。2.心肌组织线粒体呼吸功能的变化:各组平衡末呼吸控制率无显著性差异(P＞0.05);硫化氢后处理组、缺血后处理组及HMR1098拮抗组再灌注末的呼吸控制率各组间无明显差异,但明显高于缺血组以及5HD拮抗组(P＜0.05),5HD拮抗组的RCR与缺血组比较,其差异无统计学意义(P＞0.05)。3.心肌细胞线粒体膜电位:与正常组膜电位相比,复氧末H/R组、5HD组膜电位均显著下降(P＜0.05);NaHS 10μM、HMR1098组与IPTC组三组组间膜电位差异无统计学意义(P＞0.05),但HMR1098与5HD组比较,差异有统计学意义(P＜0.05)。4.F-肌动蛋白的变化:心肌细胞缺氧复氧后组间比较,正常组F-肌动蛋白的荧光强度明显弱于其余各组(P＜0.05);硫化氢后处理组以及缺氧后处理组荧光强度明显高于缺氧组(P＜0.05);硫化氢后处理组与缺氧后处理组之间差异无统计学意义(P＞0.05)。结论:1.三种浓度的硫化氢后处理均可模拟缺血后处理对离体灌注心脏的心功能产生一定的保护作用,且各浓度之间无量效关系;2.硫化氢后处理的作用位点可能是线粒体ATP敏感性钾通道,与肌膜钾通道无关;3.硫化氢后处理对缺血心肌保护作用的机制可能与稳定心肌细胞线粒体膜电位进而抑制凋亡,以及促进F-肌动蛋白聚集有关,同时也有助于线粒体呼吸功能的尽快恢复。更多还原

【Abstract】 Objective To explore the effects of hydrogen sulfide postconditioning on function and structure of rat ischemic myocardium using both Langendorff perfused rat hearts and hypoxia/reoxygenation model of adult rat cardiomyocytes.Methods and ResultsLangendorff perfused Sprague-Dawley male rat hearts were randomized into 8 groups.After 20min equilibration ischemia(40min)/reperfusion(60min)(I/R) was induced.After 40 min ischemia,rat hearts were perfused with Krebs-Henseleit buffer containing different concentrations of NaHS(1,10,100uM) for 2min,or subjected to six 10-second cycles of ischemia/reperfusion.To evalate the ATP sensitive postassium chanels in hydrogen sulfide postconditoning, 5-hydroxydecanoate(5HD),or HMR1098 was treated before 10uM NaHS.The hearts were then reperfused for 60min.Normal hearts were left untreated.Heart function was assessed by LVDP,±dp/dtmax,HR and LVEDP;mitochondrial respiratory function was assessed by respiratory control rate(RCR).Comparied with equilibration,reduction trends of heart function was found in all groups.Heart function of I/R group was significantly decreased.NaHS and ischmia postconditing preserved heart function.5HD abolished the protective effects of postconditioning while HMR1098 can imitate the postconditioning effects.At the end of reperfusion RCR was decreased in I/R group,but was protected by NaHS 10uM and ischmia postconditioning antagonizing by 5HD but not by HMR1098.Cultured cariomyocytes from the same donor rat were subjected to hypoxia/reoxygenation, After hypoxia,cells were treated with 10uM Naris or IPTC,with or without pretreatment with 5HD/HMR1098.Mitochondrial membrane potential and F-actin fluorecense were determined.At the end of reoxygenation,hypoxia/reoxygenation significantly decreased mitochondrial membrane potential,NaHS and IPTC protected the injury,5HD but not HMR1098 reversed the postconditioning effects.Hypoxia/reoxygenation significantly increased F-actin fluorescence. Fluorescence intensity of F-actin of 10uM NaHS,or IPTC were higher than hypoxia/reoxygenation.Conclusions:NaHS postconditioning improves myocardial function and mitochondrial respiratory function.This effect can be blocked by 5HD but not by HMR1098,suggesting the involvement of mitochondrial ATP sensitive potassium channel.In cultured rat cardiomyocytes,this protection is associated with preservation of mitochondrial membrane potential and aggregation of F-actin.更多还原