组织因子反义寡脱氧核苷酸防治心肌缺血再灌注损伤的实验研究

Inhibition of antisense oligodeoxynucleotide for tissue factor to myocardial ischemic-reperfusion injury

目的:研究组织因子(TF)反义寡脱氧核苷酸(AS/TF)对大鼠心肌缺血再灌注损伤的作用。方法:设计针对大鼠TF的AS/TF、正义寡脱氧核苷酸(S/TF)和错配寡脱氧核苷酸(Sc/TF)。50只雄性Wistar大鼠随机分为假手术组(Sham组)、缺血再灌注损伤组(I/R组)、反义寡脱氧核苷酸防治组(AS/TF组)、正义寡脱氧核苷酸防治组(S/TF组)和错配寡脱氧核苷酸防治组(Sc/TF组),每组10只。Sham组和I/R组大鼠经静脉注入生理盐水,AS/TF组、S/TF组和Sc/TF组大鼠分别注入AS/TF、S/TF和Sc/TF。于注射后10h麻醉大鼠,开胸暴露心脏,于冠状动脉左前降支中1/2处穿线,Sham组只穿线不结扎,其余4组均结扎造成心肌缺血,90min后解除结扎,再灌注1h。用ELISA检测大鼠血清心肌肌钙蛋白I(cTnI)、血浆凝血酶鄄抗凝血酶复合物(TAT)和血小板P鄄选择素的含量。Northern印迹杂交检测大鼠缺血区心肌组织中TF基因的转录,HE染色观察缺血区心肌组织的病理变化。结果:大鼠心肌缺血再灌注后,血清cTnI、血浆TAT和P鄄选择素含量明显上升(P<0.001),AS/TF组的上升幅度低于I/R组、S/TF组和Sc/TF组(P<0.05)。Northern印迹杂交显示大鼠缺血区心肌组织TF基因的转录明显增强,AS/TF组TFmRNA的转录弱于I/R组、S/TF组和Sc/TF组。HE染色可见大鼠缺血区心肌组织有灶性坏死。

Objective To study the effects of antisense oligodeoxynucleotide(AS/TF) for rat tissue factor (TF) on the myocardial ischemic reperfusion injury. Methods Antisense oligodeoxynucleotide (AS/TF), sense oligodeoxynucleotide (S/TF) and scrambled oligodeoxynucleotide (Sc/TF) for rat TF were designed. Fifty male Wistar rats were randomly divided into Sham group, ischemia reperfusion group (I/R group), antisense oligodeoxynucleotide administration group (AS/TF group), sense oligodeoxynucleotide administration group (S/TF group) and scrambled oligodeoxynucleotide administration group (Sc/TF group), with 10 rats in each group. Each rat was administered intravenously 4 ml/kg of saline in Sham and I/R group, and 2.8 mg/kg of AS/TF, S/TF and Sc/TF dissolved respectively in 4 ml/kg of saline in AS/TF group, S/TF group and Sc/TF group separately. After 10 hours, rats were anaesthetized with heart exposion. Anterior descending branch of left coronary artery of each rat in I/R group, AS/TF group, S/TF group and Sc/TF group was ligated to accomplish myo-cardial ischemia, and after 90-min ischemia, the heart was reperfused for 1 hour. A suture was only put without ligation under the anterior descending branch of left coronary artery in Sham group. Blood sample was collected from carotid artery before the myocardial ischemia, at the end of the ischemia and reperfusion respectively to detect cardiac troponin I (cTnI), thrombin-antithrombin complex (TAT) and the granule membrane protein 140 (GMP-140) by enzyme-linked immunoabsorbent assay(ELISA). When the reperfusion was over, the ischemic myocardial tissue RNA was isolated for detecting TF mRNA by Northern blot, and hematoxylin-eosin (H-E) staining was also performed to investigate the myocardial morphology. Results After the myocardial ischemia and reperfusion, blood concentrations of cTnI, TAT and GMP-140 increased (P<0.001, vs Sham group), which were lower in AS/TF group than those in the I/R group, S/TF group and Sc/TF group respectively (P<0.05). Northern blot analysis showed intensified TF mRNA in the ischemic and reperfused myocardial tissue, while the transcription of TF in the myocardial tissue in AS/TF group was inferior to that in I/R group, S/TF group and Sc/TF group. H-E staining showed focal necrosis, lytic myocardial fibers, scattered hemorrhage and infiltration of inflammatory cells in the ischemic and reperfused myocardial tissue. In I/R group, S/TF group and Sc/TF group, however, the myocardial injury was more severe than that in AS/TF group. Conclusions The transcription and expression of TF gene are enhanced during myocardial ischemia reperfusion, which the antisense oligodeoxynucleotide of TF can be specifically suppress the synthesis and release of TF and thereby inhibit the activation of the blood coagulation and the ischemic reperfusion injury to the rat myocardial tissue.