Netrin-1对活体大鼠心肌缺血再灌注损伤影响的实验研究

Experimental study of the effect of Netrin-1 on in vivo myocardial ischemia reperfusion injury in rats

目的研究Netrin-1对活体大鼠心肌缺血再灌注损伤(Ischemia reperfusion injury,I/R)的影响及机制。为临床上提供体外循环(Cardiopulmonary bypass,CPB)下心肌保护新的策略。方法雄性Spraghe-Dawley(SD)大鼠20只,体重450-500g,随机分成2组,体外循环预充液中添加Netrin-1(10只)[I/R+Netrin-1组],体外循环预充液中不添加Netrin-1(10只)[I/R组]。建立无血预充大鼠深低温停循环(Deep hypothermic circulatory arrest,DHCA)的心肌缺血再灌注的模型。实验结束2h后处死大鼠留取心脏标本。在CPB前、CPB75min(复温后15min),CPB后2h留取大鼠动脉血测定心肌损伤指标[心肌肌钙蛋白I(c Tn I)和心型脂肪酸结合蛋白(HFABP)]。实验结束取大鼠心脏标本用TUNEL法检测心肌细胞凋亡;用Western-blot法测定局部心肌组织直肠癌结肠癌缺失基因(Delete in colorectal carcinoma gene,DCC)蛋白指标。大体光镜下观察心脏组织病理形态学改变;大鼠CPB前、CPB 15min、CPB 45min(停循环15min)以及CPB 90min(复温30min)监测血气分析。结果 I/R+Netrin-1组较I/R组:血清中心肌肌钙蛋白I(c Tn I)和心型脂肪酸结合蛋白(HFABP)含量明显减少;心肌缺血再灌注后心肌凋亡明显减少27.5%(P<0.05);DCC表达增加(P<0.05)。HE染色镜下观察发现I/R+Netrin-1组心肌细胞损伤程度明显减轻。结论Netrin-1对心肌缺血再灌注损伤有保护作用,其机制可能与DCC有关。

Objective To observe the effect and mechanism of Netrin-1on in vivo myocardial ischemia reperfusion injury in rats. For the clinic with myocardial protection about cardiopulmonary bypass with a new strategy. Metholds Twenty male Sprague-Dawley rats,weight （400-450）g,were randomly divided into two groups（each=10）：I/R＋Netrin-1 and I/R groups. To estab-lish of myocardial ischemia reperfusion model of no blood priming of rats with deep hypothermic circulatory arrest （ DHCA）. Two groups of rats were given tracheal intubation in general anesthesia,mechanical ventilation,and cardiopulmonary bypass intubation. Blood samples were collected at the before of CPB,15min after rewarming and 2h after CPB for determination of myocardial injury markers of cardiac troponin I （cTn I） and heart fatty acid binding protein （HFABP）. These was measured by Enzyme-linked im-munosorbent assay （ELISA）. Heart samples were collected for detection of myocardial cell apoptosis with TUNEL method and de-tection of local myocardial DCC protein index with Western-blot method. hematoxylin and eosin （HE） staining of cardiac tissue specimens were observed in the Myocardium Pathological Changes. Blood samples were collected at the before of CPB ,15min after CPB and DHCA,at the termination of CPB and 2h after CPB for blood gas analysis. Results The I/R＋Netrin-1group than in I/R group：serum cardiac troponin I （cTn I） and heart fatty acid binding protein （HFABP） expression was significantly reduced;myocar-dial apoptosis after myocardial ischemia-reperfusion injury decreased 23.9% （P〈0.05）;the expression of DCC increased （P〈0.05）. He staining microscopic observations showed that I/R＋Netrin-1 group significantly reduced myocardial cell injury. At the same time,We found that I/R＋Netrin-1 group of myocardial cell injury is reduced significantly through HE staining in light microscopy observation. Conclusion Netrin-1on myocardial ischemia reperfusion injury has a protective effect, its mechanism may be related with DCC.