心肌梗死后睫状神经营养因子表达的动态过程与迷走神经支配及重构

Dynamic process of the expression of ciliary neurotrophic factor and the innervation and reconstitution of vagus nerve after myocardial infarction

目的:探讨睫状神经营养因子在心肌梗死后的表达是否具有动态过程,及其在迷走神经重构中的作用。方法:①实验于2003-08/2004-02在哈尔滨医科大学附属第一医院中心实验室完成。选用健康清洁级Wistar雄性大鼠56只,分为心肌梗死后3,7,30d取材的3个心肌梗死组及相应时间点取材的3个假手术组。心肌梗死模型的制作:将大鼠麻醉后,于左心耳与肺动脉圆锥间环绕左冠状动脉主干标志处结扎冠状动脉。假手术组为穿线后不结扎冠状动脉。②从各组大鼠梗死中心、梗死周边、室间隔和右室取材,通过原位杂交及免疫组化方法检测睫状神经营养因子的mRNA及其蛋白的表达、抗囊状乙酰胆碱转运体的免疫组化方法检测迷走神经支配,并通过图像分析测定心肌中睫状神经营养因子的阳性染色密度及迷走神经支配密度。③组间比较应用t检验,睫状神经营养因子与迷走神经支配的相关性分析采用线性相关分析。结果:术后41只大鼠存活。心肌梗死后3,7,30d组分别为7和8及8只,其余18只大鼠为各假手术组(各组均为6只)。①心肌中睫状神经营养因子mRNA的表达:假手术组中未检测到睫状神经营养因子mRNA的表达。心肌梗死后7和30d组梗死周边明显高于心肌梗死后3d组(t=2.986,3.025,P<0.01);心肌梗死后30d组室间隔明显高于心肌梗死后3d组(t=3.126,P<0.01)。②心肌中睫状神经营养因子蛋白的表达:假手术组中未检测到睫状神经营养因子蛋白的表达。仅在心肌梗后30d组的梗死周边和室间隔心肌中检测到睫状神经营养因子蛋白的表达[(9.54±2.36)%,(11.25±4.60)%]。③心肌中迷走神经支配密度:心肌梗死组中梗死中心区在心肌梗死后不同时间为0。心肌梗死后30d组梗死周边明显高于心肌梗死后3和7d组及相应假手术组(t=2.452～3.186,P<0.05～0.01)。心肌梗死后室间隔和右心室7和30d组明显高于相应假手术组(t=2.327～3.467,P<0.05～0.01);心肌梗死后30d组室间隔和右心室明显高于心肌梗死后3和7d组(t=2.506～3.332,P<0.05～0.01)。④相关性:心肌梗死后7d组梗死周边心肌细胞中睫状神经营养因子mRNA的表达与迷走神经支配呈显著正相关(r=0.51,P<0.05),心肌梗死后30d组室间隔心肌细胞中睫状神经营养因子mRNA的表达与迷走神经支配也呈显著正相关(r=0.65,P<0.05)。结论:①睫状神经营养因子可能主要在心肌梗死等病理状态下发挥一定的作用。②睫状神经营养因子可能在心肌局部发挥其神经营养作用进而参与心肌梗死后的神经重构及神经内分泌的调节过程。

AIM： To explore whether the expression of ciliary neurotrophic factor has a dynamic process or not after myocardial infarction, and its role in the reconstitution of vagus nerve. METHODS： ① The experiment was carried out in the central laboratory of the First Affiliated Hospital of Harbin Medical University from August 2003 to February 2004. Fifty-six healthy male Wistar rats of clean degree were used. They were divided in to 3 myocardial infarction groups in which the materials were taken at 3, 7 and 10 days after myocardial infarction, and 3 sham-operated groups in which the materials were taken at corresponding time points. Establishment of the models of myocardial infarction： After the rats were anesthetized, the coronary artery was ligated at the left auricle＂ and pulmonary arterialcone orbited left coronary artery main stem. In the sham-operated groups, the coronary artery was not ligated. ② In infarcted center, peri-infarcted area, septum and right ventricle, the expressions of expression of ciliary neurotrophic factor mRNA and protein were examined with in situ hybridation and immunohistochemical methods, innervation of vagal nerves was detected with vesicular acetylcholine transporters （VACHT） immunohistochemistry. The densities of expression of ciliary neurotrophic factor and the density of vagal innvervation were analyzed with Motic image-analyzing system. ③ The t test was applied in the intergroup comparison, and the linear correlation analysis was used in analyzing the correlation between ciliary neurotrophic factor and the innvervation of vagal nerves. RESULTS： 41 rats survived after ligation. There were 7, 8 and 8 rats in the 3, 7 and 30 days after myocardial infarction groups, the other 18 rats were in the shame-operated groups with 6 rats in each group. ① The expression of ciliary neurotrophic factor mRNA： It was not detected in the sham-operated groups. The expression in the peri-infarcted area was obviously higher in the 7 and 30 days after myocardial infarction groups than in the 3 days after myocardial infarction group （t=2.986,3.025,P 〈 0.01）. The expression in septum was obviously higher in the 30 days after myocardial infarction group than in the 3 days after myocardial infarction group （t=3.126, P 〈 0.01）. ② The expression of ciliary neurotrophic factor protein： It was not detected in the sham-operated groups. The expression was only detected in the peri-infarcted area and septum in the 30 days after myocardial infarction group [（9.54±2.36）%, （11.25±4.60）%]. ③ The density of vagal innervation in myocardium： It was 0 in the infarcted center at different time point after myocardial infarction in the myocardial infarction groups. In peri-infarcted area, the density of vagal innervation was obviously higher in the 30 days after myocardial infarction group than in the 3 and 7 days after myocardial infarction groups （t=2.452-3.186, P 〈 0.05-0.01）. The densities in septum and right ventricle in the 7 and 30 days after myocardial infarction groups were obviously higher than those in the corresponding sham-operated groups （t=2.327-3.467, P 〈 0.05-0.01）. The densities in septum and right ventricle were obviously higher than in the 30 days after myocardial infarction group than in the 3 and 7 days after myocardial infarction groups （t=2.506-3.332,P 〈 0.05-0.01）. ④ Correlation： In the 7 days after myocardial infarction group, the expression of ciliary neurotrophic factor mRNA in peri-infarcted myocardial cells was significantly and positively correlated with vagal innervation （r=0.51,P 〈 0.05）. In the 30 days after myocardial infarction group, the expression of ciliary neurotrophic factor mRNA in the myocardium of ventricular septum was also significantly and positively correlated with vagal innervation （r=0.65, P 〈 0.05）.CONCLUSION： ① Ciliary neurotrophic factor maybe mainly play a certain role under the pathological state of myocardial infarction. ② Ciliary neurotrophic factor might play the neuro-nutritious role in myocardium, and then take part in neural remodeling and the neuro-endocrinal regulation.