心肌及蛛网膜下腔NGF基因转染对1型糖尿病大鼠心脏损伤的保护作用

目的 探讨以重组腺相关病毒（rAAV）为载体，评价心肌点注射和蛛网膜下腔注射转导神经生长因子（NGF）基因对1型糖尿病大鼠心脏损伤的保护作用。方法 全实验包括两个子实验。实验一：将12只SPF级雄性SD大鼠采用随机数字表法分为2组（n=6）：对照组、糖尿病（DM）组，DM组大鼠通过注射链脲佐菌素（STZ）建立1型糖尿病模型，2组大鼠于注射STZ前1 d及后第1、2、4、6、8、9周测甩尾反射潜伏期，并在第9周时通过酶联免疫吸附试验（ELISA）检测各组织中的NGF、降钙素基因相关肽（CGRP）含量。实验二：24只SPF级雄性SD大鼠随机分为4组（n=6）：糖尿病心脏转染对照组（MC）组、糖尿病心脏转染（ME）组、糖尿病脊髓转染对照（SC）组、糖尿病脊髓转染（SE）组，采用2×2析因设计，4组糖尿病模型建模方法同DM组，成模后第4周，MC组和ME组以心脏点注射分别转染滴度为0.8×1013 μg/L携带绿色荧光蛋白（GFP）基因的重组腺相关病毒（rAAV9-GFP）和相同滴度的携带NGF基因的rAAV-GFP（rAAV9-NGF-GFP），各100 μL；SC组和SE组以蛛网膜下腔注射分别转染滴度为0.8×1012 μg/L的rAAV2-GFP和相同滴度的rAAV2-NGF-GFP，各25 μL。5周后，测各组大鼠心功能指标。取心肌、T1-T5段脊髓及背根神经节组织，荧光显微镜下观察GFP的表达情况；采用ELISA测各组织中的NGF、CGRP含量。结果 实验一中，与对照组相比，DM组的甩尾反射潜伏期在第4周后明显延长（P＜0.05），心肌组织中的NGF、CGRP明显下调（P＜0.05）。实验二中，心肌点注射法转染rAAV9-NGF-GFP可改善大鼠的各项心功能指标（P＜0.05），上调心肌组织中NGF、CGRP蛋白含量（P＜0.05）；且实验过程中的转染物与转染途径之间存在交互效应，两者联用改善心功能和上调心肌组织中NGF、CGRP的效果更显著（P＜0.05）。结论 心肌点注射rAAV-NGF-GFP可以更有效地提高1型糖尿病大鼠心肌组织中NGF的表达，产生更有效的心脏保护作用。

Cardioprotective effect of myocardial injection or subarachnoid injection mediated nerve growth factor gene transfection in type 1 diabetic rats

Objective To investigate the protective effect of recombinant adeno-associated virus (rAAV) mediated nerve growth factor (NGF) transfection by intramyocardial injection or subarachnoid injection in type 1 diabetic rats.Methods The whole experiment consists of two sub-experiments, experiment A:12 SPF grade male SD rats were randomly divided into control group (group control) and diabetes mellitus group (group DM, n=6). Group DM was given streptozotocin(STZ, 50 mg/kg, i.p) to induce type 1 diabetic rat model. The tail flick latencies were measured one day before STZ injection and 1, 2, 4, 6, 8 and 9 weeks after STZ injection in two groups of rats. The contents of NGF and calcitonin generelated peptide (CGRP) were measured by ELISA method. Experiment B:24 SPF grade male SD rats were randomly divided into 4 group (n=6): diabetic heart transfection control group (group MC), diabetic heart transfection group (group ME), diabetic spinal cord transfection control group (group SC) and diabetic spinal cord transfection group (group SE). The 2×2 factorial design was used in this study. The diabetic rat model was established by giving STZ (50 mg/kg, i.p) in four groups of rats. At the end of 4 weeks after injection of STZ, rats in group MC and ME were transfected with rAAV-green florescent protein (GFP) and rAAV-NGF-GFP at a titer of 0.8×1013 virus genomes (μg/L) by intramyocardial injection at 100 μL. Rats in group SC and SE were transfected with rAAV-GFP and rAAV-NGF-GFP at a titer of 0.8 × 1012 virus genomes (μg / L) by subarachnoid injection at 25 μL. After 5 weeks, the cardiac function indexes were measured in groups of rats. The expressions of GFP in myocardium, spinal cord and dorsal root ganglion of T1-T5 were observed by fluorescence microscope,and the contents of NGF and CGRP were measured by ELISA method. Results In experiment A, after 4 weeks, the tail flick latency was significantly delayed in group DM compared with that of group control (P＜0.05). The NGF and CGRP protein in myocardium were significantly decreased in group DM compared with those of group control (P＜0.05). In experiment B, both rAAV-NGF-GFP and myocardial tissue as transfection pathway significantly improved the cardiac function of rats (P＜0.05), and up-regulated the contents of NGF and CGRP protein in myocardial tissue (P＜0.05). There was an interaction between transfectants and transfection pathways during the experiment. Combination of both can improve cardiac function and up-regulate NGF and CGRP in myocardium (P＜0.05). The myocardial point injection of rAAV NGFGFP can significantly improve the cardiac function of rats (P＜0.01), and up-regulate the contents of NGF and CGRP protein in myocardial tissue (P＜0.01). Conclusion Compared with subarachnoid injection, intramyocardial injection of rAAV-NGF can effectively increase the expression of NGF in myocardial tissue of diabetic rats and have a more effective cardioprotective effect.