国际麻醉学与复苏杂志   2018, Issue (6): 1-1
    
心肌降钙素基因相关肽基因转导对糖尿病大鼠心肌缺血/再灌注损伤的保护作用
王玥, 岳维, 李兔平, 高盼, 易剑敏, 张伟男1()
1.山西医科大学麻醉学系
Protective effect of myocardial calcitonin gene-related peptide gene transduction on myocardial ischemia/reperfusion injury in diabetic rats
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摘要:

目的 评估心肌点注射法慢病毒(lentivirus, Lv)介导降钙素基因相关肽(calcitonin gene?蛳related peptide, CGRP)基因转导糖尿病大鼠的可行性及此法对糖尿病心肌在遭受缺血/再灌注(ischemia/reperfusion, I/R)损伤时是否具有保护作用。 方法 将60只200~220 g健康雄性SD大鼠采用随机数字表法分为5组(每组12只):对照组(C组)、糖尿病组(DM组)、心肌注射手术组(Sham组)、心肌注射对照组(MC组)、心脏转导组(ME组)。采用心肌点注射法将Sham组、MC组、ME组大鼠在体分别注射生理盐水、滴度2×107病毒基因组(virus genomes, vg)/ml的携带增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)基因的慢病毒(Lv?蛳eGFP)及携带CGRP基因的Lv?蛳eGFP(Lv?蛳CGRP?蛳eGFP)各 50 μl。转染1周后DM组、Sham组、MC组、ME组行链脲佐菌素(streptozocin, STZ)50 mg/kg腹腔注射,建立糖尿病模型。注射STZ 8周后采用冠状动脉左前降支结扎法制备心脏I/R模型。比较各组大鼠甩尾反射潜伏期,心肌、脊髓、背根神经节(dorsal root ganglion, DRG)组织中eGFP表达情况、CGRP基因转录水平、CGRP蛋白表达含量,心肌缺血危险区面积(area at risk, AAR)及梗死区面积(infarct size, IS)。 结果 与C组比较,DM组、Sham组、MC组、ME组大鼠自糖尿病造模成功4周末开始甩尾反射潜伏期明显延长(P<0.05)。DM组、Sham组、MC组缺血区心肌、T1~T5脊髓、 T1~T5 DRG CGRP转录水平低于C组、ME组(P<0.05), ME组缺血区心肌、T1~T5 DRG CGRP转录水平高于C组(P<0.05)。C组、ME组T1~T5脊髓、T1~T5 DRG、缺血区心肌及血清CGRP含量高于DM组、Sham组、MC组(P<0.05),ME组T1~T5脊髓、缺血区心肌CGRP含量高于C组(P<0.05)。DM组、Sham组、MC组IS/AAR高于C组、ME组(P<0.05),ME组IS/AAR高于C组(P<0.05)。 结论 心肌点注射转导CGRP基因可有效提高糖尿病大鼠心脏I/R时CGRP表达,CGRP增多可减轻糖尿病大鼠心脏I/R损伤。

关键词: 降钙素基因相关肽; 糖尿病; 心肌; 缺血/再灌注
Abstract:

Objective To evaluate the feasibility of lentivirus (Lv) mediated calcitonin gene-related peptide(CGRP) gene transduction in diabetic rats by myocardial injection and whether this method has a protective effect on diabetic myocardium during ischemia/reperfusion(I/R) injury. Methods Sixty healthy male SD rats, weighing 200-220 g, were randomly divided into control group (group C), diabetes mellitus group (group DM), Sham transduction group (group Sham), myocardial injection control group (group MC) and myocardial injection experiment group(group ME), in accordance with a random number table. Rats in group Sham, group MC and group ME were given with normal saline, at a titer of 2×107 virus genomes(vg)/ml Lv-enhanced green fluorescent protein(eGFP) and Lv-CGRP-eGFP 50 μl respectively by myocardial injection. One week later, rats in group DM, group Sham, group MC and group ME received streptozocin(STZ) 50 mg/kg intraperitoneal injection to induce diabetes. Eight weeks after the injection of STZ, myocardial I/R was induced by occlusion and release of left anterior descending branch of coronary artery. The latency of tail flick reflection, the expression of eGFP, the transcription level of CGRP, the expression of CGRP in myocardium, spinal cord and dorsal root ganglion(DRG) and myocardial ischemia area, infarct size(IS) area were measured. Results Compared with group C, the latency of tail flick reflection in group DM, group Sham, group MC and group ME were significantly prolonged in the 4th week after diabetic model successfully manufactured(P<0.05). The transcription levels of CGRP in the ischemic myocardium, spinal cord T1-T5 and DRG T1-T5 in group DM, group Sham and group MC were lower than those in group C and group ME (P<0.05). The level of CGRP in ischemic myocardium and DRG T1-T5 of group ME was higher than that in group C(P<0.05). The content of CGRP in cord T1-T5, DRG T1-T5, ischemic myocardium and serum in group C and group ME were higher than those in group DM, group Sham and group MC (P<0.05). The content of CGRP in cord T1-T5, ischemic myocardium in group ME was higher than that in group C (P<0.05). The infarct size in group DM, group Sham, group MC were higher than that in group C and group ME(P<0.05), the infarct size of group ME was higher than that in group C(P<0.05). Conclusions Intramyocardial injection of CGRP gene can effectively enhance the expression of CGRP at I/R of diabetic rat′ heart. Increased CGRP may reduce cardiac I/R injury in diabetic rats.

Key words: Calcitonin gene-related peptide; Diabetes mellitus; Myocardium; Ischemia-reperfusion