大鼠心肌解剖无复流现象中中性粒细胞的浸润和GDF-15的动态表达

Neutrophil infiltration and GDF-15 expression in rat myocardial anatomic no-reflow phenomenon

目的:研究大鼠心肌解剖无复流现象中中性粒细胞的浸润和生长分化因子15(growth differentiation factor 15,GDF-15)的动态表达。方法:雄性Wistar大鼠144只,随机分为手术组(I/R组)和假手术组(sham组)。手术组用Y形线结扎冠状动脉左前降支,缺血60 min,再灌注分别2 h、4 h、6 h、12 h、24 h和7 d。假手术组只穿线不结扎。分别用硫磺素S、酞菁蓝和氯化三苯基四氮唑(TTC)染色评估大鼠心肌解剖无复流面积及梗死面面积;real-time PCR法测定GDF-15 mRNA表达水平;免疫组化法测定GDF-15蛋白表达;通过检测髓过氧化物酶(myeloperoxidase,MPO)活性间接检测中性粒细胞浸润量,ELISA酶联免疫法检测心肌组织匀浆中MPO活性;HE染色观察镜下心肌损伤及中性粒细胞浸润情况。结果:I/R组无复流面积在再灌注2～6 h期间内延展最快,sham组无缺血及无复流现象发生;I/R组各时点心肌组织中GDF-15表达量和MPO活性均较sham组明显增高,且呈规律性变化。在再灌注2～24 h之间,MPO活性随GDF-15表达增高而降低,二者呈负相关(r=-0.9895)。结论:无复流现象中同时存在中性粒细胞的浸润和GDF-15的表达,且二者变化呈负相关,推测GDF-15可能通过抑制中性粒细胞的浸润而抑制无复流的延展。

AIM： To investigate the neutrophil infiltration and the expression of growth differentiation factor 15 （GDF-15） in rat myocardial anatomic no-reflow phenomenon. METHODS： Male Wistar rats were randomly divided into operation group （I/R group） and sham operation group （sham group）. Myocardial ischemia was induced by the ligation of left anterior descending coronary artery with the Y-shaped sutures, and the rats were subject to ischemia for 60 min followed by reperfusion for 2 h, 4 h, 6 h, 12 h, 24 h or 7 d. The rats in sham group did not undergo occlusion of the coronary arter- y. The mRNA expression of GDF-15 was measured by real-time PCR. The protein level of GDF-15 was determined by im- munohistochemical analysis. The activity of myeloperoxidase （MPO） in the myocardial tissues was measured by ELISA. The areas of myocardial anatomy no-reflow and infarction were assessed by thioflavin S, phthalocyanine blue and tripheny- ltetrazolium chloride staining. The cardiac histological infiltration of neutrophils was assessed by hematoxylin-eosin stai- ning. RESULTS ： The fastest extension of the no-reflow areas was formed from 2 h to 6 h of reperfusion in I/R group. Is- chemia and no-reflow phenomenon didn＇ t happen in sham group. In the myocardial tissues, the expression of GDF-15 and the activity of MPO in I/R group were higher than those in sham group at each time point. The activity of MPO deceased with high expression of GDF 15 （r = -0. 9895）. CONCLUSION： Neutrophil infiltration and the expression of GDF-15 exist in the no-reflow myocardium with a negative correlation between them. GDF-15 can antagonize the accumulation and infiltration of neutrophils, and also inhibit the extension of no-reflow area.