rAAV9-NGF基因转染糖尿病大鼠对心脏损伤的保护作用

Cardioprotective effect of recombinant adeno-associated virus mediated nerve growth factor gene transfection in diabetic rats

目的探讨通过心肌点注射,重组腺相关病毒9型(recombinant adeno-associated virus serotype 9,rAAV9)介导神经生长因子(nerve growth factor,NGF)基因转染糖尿病大鼠,对心脏损伤的保护作用。方法大鼠糖尿病模型建立后4周,心脏点注射携带NGF基因的重组腺相关病毒(rAAV9-NGF),使心肌组织过表达NGF。5周后,免疫荧光检测NGF、降钙素基因相关肽(calcitonin gene-related peptide,CGRP)和神经纤维的分布;血流动力学参数评估心脏功能;ELISA法检测心肌组织中NGF、 CGRP等蛋白表达水平。结果 rAAV9介导的外源基因NGF可稳定转染,且过表达于糖尿病大鼠的心肌组织,LVSP、HR、+dp/dt_(max)明显下降,LVEDP、-dp/dtmax明显上升,心功能改善;心肌组织中的NGF、CGRP蛋白上调;免疫荧光结果显示,转染后可部分逆转心肌组织NGF、CGRP和神经纤维的减少。结论心肌点注射rA AV9-NGF基因可有效提高糖尿病大鼠心肌NGF的表达,促进神经纤维的生长,产生对心脏的保护作用。

Aim To investigate the feasibility of recombinant adeno-associated virus serotype9(rAAV9)mediated nerve growth factor(NGF)transfection in diabetic rats by intramyocardial injection,and to confirm the protective effect of this method on diabetic cardiac autonomic neuropathy.Methods At 4th week after the establishment of diabetic model,recombinant adeno-associated virus(rAAV9-NGF)carrying NGF gene was injected into the heart of rats.Five weeks later,the distribution of NGF,calcitonin gene-related peptide(calcitonin gene-related peptide,CGRP)and nerve fibers were detected by immunofluorescence.Hemodynamic parameters were used to evaluate cardiac function,and ELISA method was used to detect the expression of NGF,CGRP in myocardium.Results The exogenous gene NGF mediated by rAAV9 could be stably transfected and over-expressed in the myocardium of diabetic rats.The left ventricular systolic pressure,heart rate,and maximal rate of increase of left ventricular pressure significantly decreased,the left ventricular end-diastolic pressure and the maximal decrease rate of left ventricular pressure significantly increased,and the cardiac function was improved.NGF,CGRP protein in myocardial tissues were up-regulated,and immunofluorescence showed that the decrease of NGF,CGRP and nerve fibers in myocardial tissues could be partially reversed after transfection.Conclusions Intramyocardial injection of NGF gene can effectively enhance the expression of NGF and promote the growth of nerve fiber,exerting a protective effect in diabetic rat heart.