摘要：
目的 观察神经生长因子 (NGF) 基因转染对糖尿病大鼠在体心脏缺血-再灌注 (IR) 损伤的影响.方法 健康雄性SD大鼠腹腔注射链脲佐菌素50 mg·kg-1制备糖尿病模型, 2周后造模成功的28只糖尿病大鼠随机分为糖尿病对照组、模型组、载体组和NGF组 (n=7), 采用心肌点注射基因转染方法, 载体组和NGF组分别注射包被绿色荧光蛋白的腺相关病毒9 (AAV9-GFP) 和携带NGF的AAV9-GFP (滴度均为8×1014 vg·L-1) 各100μL.基因转染4周后, 模型组、载体组和NGF组大鼠结扎左冠状动脉前降支缺血30 min再灌注120 min制备IR损伤模型.利用电生理信号记录仪监测缺血前即刻 (T0) 和再灌注120 min末 (T1) 大鼠左室舒张末压 (LVEDP) 、左室收缩压 (LVSP) 、左心室内压最大上升和最大下降速率 (±dp/dtmax) 及心率 (HR) .免疫荧光法和ELISA法观察NGF、降钙素基因相关肽 (CGRP) 表达水平和心肌神经纤维分布.结果 T0时, 糖尿病对照组、模型组和载体组大鼠心功能各指标均无显著差异 (P＞ 0.05);与模型组和载体组比较, NGF组大鼠LVSP和HR显著升高, LVEDP降低 (P ＜0.05), 而±dp/dtmax无显著差异 (P＞ 0.05) .与T0时相比, T1时除糖尿病对照组外, 模型组、载体组和NGF组LVSP、HR和±dp/dtmax均显著降低, LVEDP显著升高 (P ＜0.05) .与糖尿病对照组比较, 模型组大鼠LVSP、 HR和±dp/dtmax均显著下降, LVEDP显著升高 (P ＜0.05), NGF、 CGRP表达量显著上调 (P ＜0.05), 心肌神经纤维分布减少, 载体组与模型组各指标无显著差异 (P＞ 0.05);与模型组和载体组比较, NGF组大鼠LVSP、 HR显著升高, LVEDP降低, 心肌NGF、 CGRP蛋白表达上调 (P ＜0.05), 神经纤维分布有所增多.结论 NGF基因转染可减轻糖尿病大鼠心脏IR损伤, 可能与NGF和CGRP增加有关.%AIM To observe the ef fects of nerve growth factor (NGF) gene transfection on ischemia-reperfusion (IR) injury of diabetic rat heart in vivo. METHODS Healthy male SD rats were injected intraperitoneally with streptozotocin 50 mg·kg-1 to prepare diabetes model. After 2 weeks of modeling, 28 diabetic rats were randomly divided into diabetic control group, model group, vector group and NGF group (n =7). Myocardial point injection gene transfection method was adopted. Vector group and NGF group were injected with green fluorescent protein-containing adeno-associated virus 9 (AAV9-GFP) and AAV9-GFP carrying NGF (AAV9-NGF-GFP) 100 μL (titer 8 × 1014 vg·L-1), respectively. Four weeks after gene transfection, IR injury model was prepared in the model group, the vector group and the NGF group by ligation of left anterior descending coronary artery ischemia for 30 min and reperfusion for 120 min. Left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure rise and drop (±dp/dtmax) and heart rate (HR) were collected using electrophysiological signal recorders immediately before ischemia (T0) and 120 min after reperfusion (T1). Immunofluorescence and ELISA were used to analyze the changes of NGF, calcitonin gene-related peptide (CGRP) and myocardial nerve fiber distribution in myocardial tissue. RESULTS At T0, there were no significant differences in cardiac function among the diabetic control group, the model group and the vector group (P＞ 0.05). Compared with the model group and the vector group, LVSP and HR were significantly increased, LVEDP was decreased (P ＜ 0.05), but the differences of±dp/dtmax were not significant (P＞ 0.05) in the NGF group. At T1, compared with T0, LVSP, HR and ±dp/dtmax were significantly decreased and LVEDP was significantly increased in the model group, the vector group and the NGF group (P ＜ 0.05). Compared with the diabetic control group, LVSP, HR and ±dp/dtmax were decreased significantly, and LVEDP was significantly increased in the model group (P ＜ 0.05), NGF and CGRP expression levels were significantly up-regulated (P ＜ 0.05) and the distribution of myocardial nerve fibers was decreased. There were no significant difference between the vector group and the model group (P＞ 0.05).Compared with the model group and the vector group, the LVSP and HR in the NGF group were significantly increased, the LVEDP was significantly decreased, and the myocardial NGF and CGRP protein expression were significantly up-regulated (P ＜ 0.05), the distribution of nerve fibers was increased. CONCLUSION NGF transfection can alleviate myocardial IR injury in diabetic rats, which may be related to the increase of NGF and CGRP.
