RESUMO
Aim To investigate the effects of lentivirus mediated nerve growth factor ( NGF) gene silencing on pheochromocytoma cells ( PC12 ) and the possible mechanisms .Methods The NGF shRNA expression vector was constructed .PC12 cells were randomly divi-ded into five groups (n=3 each) as follows: negative control group ( NC ) , control lentivirus group ( LV CON) , lentivirus NGF shRNA1 group ( LV shNGF1 ) , lentivirus NGF shRNA2 group(LV shNGF2), lentivir-us NGF shRNA3 group(LV shNGF3).The cells in NC group were cultured in DMEM/HG and polybrene me-dium, while others were cultured in DMEM/HG, poly-brene and corresponding lentivirus medium .After the treatment, the infection efficiency was determined by fluorescent microscope .Relative expression of NGF , extracellular signal-regulated kinase ( ERK1/2 ) and p-ERK1/2 were assessed by Western blot .The expres-sion of NGF mRNA was analyzed by quantitative re-verse transcription polymerase chain reaction ( qRT-PCR) .The differentiation degree was valued according to the length of neuritis and max diameter of cells .The cell viability was detected by CCK-8.Results The in-fection efficiency in PC12 cells reached over 90%. Compared with NC group , the relative expression of NGF mRNA and NGF protein was significantly down-regulated ( P<0.05 ) .There was no difference in the expression of ERK1/2 protein and cell viability .The expression of p-ERK1/2 protein was markedly down-regulated in LV shNGF3 group ( P<0.01 ) .The cells morphology was changed , and the length of neuritis and max diameter of cells were strained in LV shNGF 3 group than those in NC group ( P<0.01 ) .Conclusion Lentivirus-mediated NGF gene silencing inhibits the differentiation of PC12 cells through suppressing the activation of ERK1/2.
RESUMO
Objective To investigate the effect of intrathecal morphine preconditioning (ITMP) on the excitability of substantia gelatinosa (SG) neurons in the dorsal horn of the spinal cord in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),group I/R,and group ITMP.Myocardial I/R injury was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.In group ITMP,the rats received intrathecal morphine 3 μg/kg (10 μl) by three cycles of 5 min infusions interspersed with 5 min infusion-free periods starting from 30 min before ischemia,and the equal volume of normal saline was given instead of morphine in group I/R.At 10 min of reperfusion,6 rats randomly selected in each group were sacrificed,and the T2-6 segments of the spinal cords were acutely isolated to prepare spinal cord slices.The resting potential,threshold of action potential (APT),peak of action potential (APP) and action potential duration in SG neurons in the dorsal horn of spinal cord slices were determined using the whole-cell patch-clamp technique,and the number of action potentials evoked by currents of 40,60,80 and 100 pA was recorded.At 120 min of reperfusion,6 rats randomly selected in each group were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size (IS) and area at risk (AAR),and IS/AAR ratio was calculated.The expression of c-fos in the T2-5 dorsal horns of the spinal cords was detected by Western blot.Results Compared with group S,the IS/AAR ratio was significantly increased,the expression of c-fos was up-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was increased,APT was decreased,and APP was increased in group I/R (P＜0.05).Compared with group I/R,the IS/AAR ratio was significantly decreased,the expression of c-fos was down-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was decreased,APT was increased,and APP was decreased in group ITMP (P＜0.05).Conclusion The mechanism by which ITMP attenuates myocardial I/R injury is related to decrease in the excitability of SG neurons in the dorsal horn of the spinal cord and reduction of responses to nociceptive stimuli in rats.