| Migraine which is serious health hazard to patients, is a common and frequently-occurring disease,with a variety of pathogenesis theories. At present there are kinds of drugs for the treatment of migraine, but the treatment efficiency is not ldeal. So searching for drugs having effects on the key signaling pathway or related targets regulation of migraine to improve the effectiveness of migraine treatment, has become one of today's medical research focus.This study mainly detected the expression of N-methyl-D-aspartate receptor (N-methyl-D-aspartate receptor, NMDAR) NR2B subunit in the trigeminal ganglia of migraine rats, to explore its function in the pathogenesis of migraine and their relationship. We down regulated the expression of PTEN gene in the trigeminal ganglion of migraine rat model with the Adenovirus AdR-siPTEN, Rat model of migraine trigeminal ganglion and investigate the impact on the behavior of migraine rats, the regulation on the expression of NO induced through NR2B signaling, Then find the possibilities of preventing and treating migraine on theory with PTEN siRNA adenovirus. Experimental method:1. Establishing nitroglycerin-type experimental migrainerat model:Sprague-Dswley (SD) were randomly divided into the control group and model group. In the control group, we injected saline instead of nitroglycerin; The model group were further divided into model components 1 hr, 4 hr, 8 hr, 12 hr, 24-hour group, subcutaneous injection of nitroglycerin injection 10mg/kg. After the rats were killed, we gained the trigeminal ganglia organizations.2. The expression of NR2B mRNA and protein in the trigeminal ganglion tissue were detected by RT-PCR semi-quantitative method and immunohistochemistry, respectively.3. SD rats were randomly divided into sham operation group, model group, nitroglycerin (GTN), AdR-siPTEN group (AdR-siPTEN + GTN), empty adenovirus group (adenovirus preservation solution + GTN, Ad-RFP control group). After AdR-siPTEN successful transfected into the trigeminal ganglion, 10mg/kg nitroglycerin was subcutaneous injected to the other three groups expect sham group. Observed the behavioral changes of each rats, which were killed 4 h later and obtained the Trigeminal ganglia tissue to detect the mRNA and protein of related genes using RT-PCR and Western blotting.4. The expression of NOS, NO in trigeminal ganglia organization of each group was measured with Nitric oxide synthase (NOS) assay kit, nitric oxide assay kit.Main findings and conclusions:1. Compared with the control group, mRNA and protein expression ofNR2B gene in trigeminal ganglion in model group changed at different time points. In the 1h model group, the expression of NR2B gene began to increase, there are significant differences in 4 hours (P <0.05), reaching a peak, and then decreased, the expression decreased to near normal 24h later, with no significant differences(P>0.05). These data proved that NR2B was involved in the development and progression of migraine.2. Red fluorescent protein expression was observed in the rats of trigeminal ganglia tissue, as the adenovirus transfected one week.RT-PCR results showed that there was no significantly difference between the sham group and the GTN group on the expression of PTEN mRNA(P>0.05) , and no significantly difference was observed in Ad-RFP control group and the GTN group, too(P>0.05). PTEN mRNA expression of AdR-siPTEN group was as much as 53.16% of Ad-RFP control group, having significant differences (P<0.05). Western blot showed that the expression of PTEN protein has no significant difference between the sham group and the GTN group (P>0.05), while there are significant difference between AdR-siPTEN and Ad-RFP, sham group(P<0.05)which indicating that through RNA interference, the expression of PTEN gene in trigeminal ganglion of migraine rat was effectively inhibited.3. With the down-regulation of PTEN gene, the behaviors of ratsscratching and climbing the cage caused by migraine was effectively relied. RT-PCR results showed that compared with the sham group , NR2B mRNA expression was significantly increased in GTN group (P <0.05) , and Ad-RFP control group has a much higher level compared to the sham group(P <0.05). However, there was no significant difference between AdR-siPTEN group and the sham group(P> 0.05), AdR-siPTEN with the GTN group , NR2B mRNA expression decreased significantly (P <0.05). compared with Ad-RFP group, NR2B mRNA expression in AdR-siPTEN group was significantly decreased (P <0.05); Western blot showed that compared with the sham group, The NR2B protein expression in Ad-RFP control group and GTN group was significantly increased, the difference was significant (P <0.05).While compared with the GTN group and Ad-RFP control group, the expression of NR2B protein decreased seriously(P <0.05).Meanwhile, siPTEN could significantly decreased the content level of NOS, NO in the trigeminal ganglia organization as compared with GTN group and Ad-RFP group(P<0.05). All these proved that down-regulation of PTEN gene has some protective effects for migraine rats. |
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