| Background and Objective:Temporal lobe epilepsy(TLE) is a common type of refractory epilepsy. At present, studys show that the important reason of refractory epilepsy is reconstruction of neural circuit loop and abnormal excitability of neural network. But its concrete pathogenic mechanisms are still poorly understood up to now.Therefore, exploration of neural network abnormal excitability is of great significance. As we know,excitatory glutamatergic neurotransmission has been considered to be related with the pathogenesis of epilepsy, including that dilivery of presynaptic neurotransmitter; dysmetabolism of glutamate in synaptic cleft; sensitivity of postsynaptic receptor. In the past, study of postsynaptic regulatory mechanism is rare. Postsynaptic density(PSD) is considered to be a tightly packed protein complex.As the important material foundation of postsynaptic regulatory mechanism, PSD can change downstream effects of neural signal transmission by regulating sensitivity of postsynaptic receptor. So this may be a core of abnormal excitability of neural network,which need to be elucidated. At present, research reports about PSD and epilepsy are not often seen.To explore the mechnism of PSD in TLE, we will first construct 2D electrophoregram of PSD fractions by use of high-throughput and high-sensitive proteomic techniques.Subsequently, PSD proteins related with temporal lobe epilepsy will be screened out and identified,which can provide new cue for further studying the relationship of PSD and TLE,and find new target for preventing and treating TLE.Methods:In this study,the Sprague-Dawley rats were randomly enrolled in SRS group,NSRS group,Normal group.The lithium-pilocarpine model of epilepsy was established. PSD fractions were gradually obtained by using sucrose gradient centrifugation and membrane sequence abstraction. we respectively isolated PSD proteins of SD rats from three groups by using two-dimensional gel electrophoresis.Then we analyzed some differential protein spots in three groups,and identified 26 proteins using MALDI-TOF-MS. In addition, the expression of three identified PSD proteins was detected in the PSD fractions by western blotting.Results:1. The lithium-pilocarpine modle of TLE: Status epilepticus were induced at the achievement ratio of 91.4%.The achivement ratio of the TLE animal model is 71.4%.The mortality rate is 20%.2. The analysis of PSD purity: Two postsynaptic mark molecules NMDA receptorl and PSD-95 were expressed in P2,synaptosome and PSD fractions,but synaptophysin(presynaptic mark) was not expressed in PSD. 3. Two-dimensional gel electrophoresis of PSD: We analyze these 2-DE images by PDQuest software.A total of 720±17 protein spots were detected in the two-PSD fraction,and a total of 286±25 protein spots were detected in the one-PSD.There is a significant difference between the two fractions(P<0.01). Compared with normal group, the expressive levels of 12 proteins were upregulated and 58 proteins were downregulated in SRS; the expression levels of 15 proteins were upregulated and 38 proteins were downregulated in NSRS.In addition,we found that there were 4 proteins only in SRS group.Compared with NSRS,the expressive levels of 15 proteins were upregulated and 25 proteins were downregulated in SRS.4. The mass spectrographic analysis of serveral differential PSD proteins: The 26 differential protein spots were identified by MALDI-TOF-MS. And these proteins consisted of members of proteins with diverse functions, including proteins related to cytoskeletal,transport,signal transduction,chaperone,energy metabolism.5. Three identified proteins (HSP27,SNX3,tubulin-alpha) were detected in PSD fractions by western blotting. The results were consistent with that of proteomic analysis and identify. The expressive levels of three proteins in three groups showed as follows, HSP27 : SRS>NSRS>Norm ; tubulin-alpha : SRS |
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