| Objective:1、Todeveloparatmodelofdiabetic peripheral neuropathyby intraperitoneal injection of streptozotocin（STZ,50mg/kg）to investigate the expression of nerve growth factor、calcitonin gene-related peptidecardiacand the decrease of nerve fibers in the heart of diabetic peripheral neuropathy rats.2、The purpose of this study was to investigate the effects of different transfection pathways on the expression of NGF gene into diabetic rats by measuring the content of NGF protein in myocardium and subarachnoid injection,respectively,and comparing the effects of different transfection pathways on the expression of NGF in the myocardium of diabetic rats.The effectiveness of the two transfection pathways was compared.3、Recombinant adeno-associated virus type 9 was used as avctor to transduct NGF gene by myocardial injection,to evaluate the feasibility of the cardioprotective effects on diabetic peripheral neuropathy rats,and the changes of nerve growth factor（NGF）,calcitomin gene-related peptide（CGRP）expression and nerve fiber regeneration in heart tissue.Methods:The study was carried out in a series of experimens:1、The establishment of diabetic peripheral neuropathy model.16 healthy adult male SD rats,weighing 200-230g,7-8weeks old,were randomly divided into control group（group control,n=8）and diabetic group（group DM,n=8）,in accordance with a random number table.The first weekend,the group DM,was given streptozotocin（STZ50 mg/kg,i.p）to induce diabetic.And fasting blood glucose was measured 24 hours after the administration,Fasting blood glucose value was higher than 16.7mmol/L for 7 times that was successfully established as diabetic rats.In the first weekend（before STZ injection）and the 10th weekend（after STZ injection）,collected the venous bloods from rats,measured the insulin concentration by enzyme-linked immunosorbent assay（ELISA）method;two groups of rats measured the blood glucose,body weight and tail-flick latency（TFL）once a week,and compared the changes of the two group with time.The expression of NGF、CGRP protein in myocardium,were measured and analyzed by enzyme-linked immunosorbent assay（ELISA）.2、Comparison of effectiveness of NGF gene transfection in myocardium and subarachnoid space of diabetic rats.36 SPF grade male SD rats,weighing200-230g,7weeks old,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）,diabetic spinal cord transfection group（group SE）.The first weekend,all of the rats was given streptozotocin（STZ50 mg/kg,i.p）to induce diabetic.At the end of 4 weeks after injection of STZ,rats in group MC and ME,were transfected with rAAV-green flurescent protein（GFP）and rAAV-NGF-GFP at a titer of 0.8×1012 virus genomes（vg/L）by intramyocardial injection at 100μl;rats in group SC and SE,were transfected with rAAV-GFP and rAAV-NGF-GFP at a titer of 0.8×1011 virus genomes（vg/L）by subarachnoid injectionl,at 25μl.5 weeks later,the expression of GFP in myocardium,T1-T5 spinal cord and dorsal root ganglia was observed under fluorescence microscope,and the content of NGF in each tissue was measured by ELISA.3、Experimental study of myocardial NGF gene transduction on cardioprotective effect in diabetic peripheral neuropathy rats.32 healthy adult male SD rats,weighing 200-230g,7-8weeks old,were randomly divided into control group（group control,n=8）,diabetic operation group（group D+P,n=8）,diabetic myocardial injection control group（group D+V,n=8）,diabetic myocardial injection experiment group（group D+NGF,n=8）,in accordance with a random number table.The latter three groups,was given streptozotocin（STZ50 mg/kg,i.p）to induce diabetic.At the end of 4 weeks after injection of STZ,rats in group control and D+P,were transfected with phosphate buffered saline（PBS）,rats in group D+V and D+NGF were transfected with rAAV9-green flurescent protein（GFP）and rAAV9-NGF-GFP at a titer of 0.8×1012 virus genomes（vg/ml）by intramyocardial injection.At the end of 5 weeks after myocardial injection,monitored the cardiac function index:left ventricular systolic pressure（LVSP）,left ventricular end-diastolic pressure（LVEDP）,heart rate（HR）,maximum rate of rise in left ventricular pressure（+dp/dtmax）,maximum rate of fall in left ventricular pressure（-dp/dtmax）histological immunofluorescence assay and ELISA were performed to evaluate the change of NGF,CGRP and never fibers.The rest is the same as the first part.Results:1、After injection of STZ,compared with the group control,the weight of rats in group DM significantly decreased（P<0.05）,the blood glucose of rats in group DM significantly increase（P<0.05）,the insulin secretion’secretion of rats in group DM significantly decrease（P<0.05）,the expression of NGF、CGRP protein in myocardium of rats in group DM significantly decrease（P<0.05）,the TFL of rats in group DM significantly increase 5 weeks later the injection of STZ（P<0.05）.The DM rats have been developed the peripheral neuropathy.2、rAAV-mediated NGF gene was stably transfected and overexpressed in myocardium,spinal cord and dorsal root ganglion of diabetic rats.Compared with MC、SC and SE group,NGF protein in me group was up-regulated significantly（P<0.01）.3、TransductionofNGFgenebyMyocardialPointInjection.Compared with the group control,the expression of NGF、CGRP protein in myocardium of rats in group D+P and group D+V significantly decrease（P<0.05）,the cardiac function index about LVSP、HR、+dp/dtmax of rats in group D+P and group D+V significantly decrease（P<0.05）,the cardiac function index about LVDEP and-dp/dtmax of rats in group D+P and group D+V significantly increase（P<0.05）.Compared with the group D+NGF,the expression of NGF、CGRP protein in myocardium of rats in group D+P and group D+V significantly decrease（P<0.05）,the cardiac function index about LVSP、HR、+dp/dtmax of rats in group D+P and group D+V significantly decrease（P<0.05）,the cardiac function index about LVDEP and-dp/dtmax of rats in group D+P and group D+V significantly increase（P<0.05）.There was no statistical difference between group control and group D+NGF（P>0.05）.The results of immunofluorescence showed:compared with the group control,the distribution of NGF,CGRP and nerve fibers in myocardium of rats in group D+P and group D+V decreased,but after transfection rAAV9-NGF,compared with group D+P and D+V,the distribution in myocardium of NGF,CGRP and nerve fibers were increased in group D+NGF.Conclusion:1、Intraperitoneal injection of high dose of STZ could effectively establish diabetic rat model,and with the prolongation of the course of disease,the thermal pain area increased and peripheral neuropathy occurred in diabetic rats.2、Compared with normal rats,endogenous NGF in myocardial tissue of diabetic rats was significantly reduced,cardiac function deteriorated and nerve fiber hyperplasia was poor in diabetic rats,which may be related to the down-regulation of CGRP secreted by sensory neuropeptides in diabetic rats.3、Compared with subarachnoid injection,myocardial injection of rAAAV-NGF could effectively increase the expression of NGF in the myocardium of diabetic rats.4、Recombinant adeno-associated virus mediated NGF gene transfection in diabetic rats can effectively increase the expression of NGF in myocardium,promote the growth of nerve fibers and protect the heart in diabetic rats.It may be related to the upregulation of CGRP secreted by sensory neuropeptide due to the increase of NGF,but the specific mechanism needs further study. |
