| Purpose:Epilepsy is a chronic recurrent brain dysfunction syndrome caused by abnormal neuron discharge, and is one of the common nervous system diseases. It is associated with marked neurodegeneration in the hippocampus, the amygdala, the entorhinal cortex, as well as the midline thalamic nuclei, which include neuron loss, axonal and/or dendritic plasticity, dentate granule cell neurogenesis, gliosis, remodel of neuronal connection, and synaptic reorganization. Temporal lobe epilepsy is the most common type of refractory epilepsy.Recent studies have identified a large number of dynamically regulated genes in several models of epilepsy by using in situ hybridization, immunohistochemistry, and cDNA microarray, which are related to immune response, inflammation, synaptic plasticity, signaling pathway, transcriptional regulator activity and cell cycle control. Although traditional hybridization methods have been successful in some cases, they are not well suited for the identification of rare messages. Compared to conventional methodologies, suppression subtractive hybridization (SSH) is a powerful technique to identify rare messages. Moreover, SSH is also a quick method characterized with low incidence of false-positives, sensitive, efficient and good reproducibility.In an effort to understand the molecule mechanism and characterize genes specifically regulated in epilepsy, SSH has been applied in this experiment in which epileptic rats were induced by lithium-chloride pilocarpine (LiCI-PILO).Methods1. Set up the rats model of TLE,14adult male Wistar rats weighing200-250g which were approved by the Shandong University Animal Center were used in this study. Rats were randomly divided into epileptic group (n=8) and control group (n=6). First,8epileptic rats were received intraperitoneal (i.p.) injection of lithium chloride (127mg/kg). Twenty four hours later, methylscopolamine (1mg/kg) was injected i.p. in order to reduce the peripheral cholinergic effects. Then after15～30min, pilocarpine (30mg/kg) was injected to counteract the peripheral cholinergic effects. The rats were injected with diazepam (10mg/kg)90min after the onset of status epilepticus to terminate the seizure. Six control rats were injected a comparable volume of sterile saline (0.9%NaCl).2. Total RNA was obtained from hippocampus of normal rats and epileptic rats. SSH was used for isolating the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pMD-19vector to set up subtractive library which was amplified through E.coli transformation, then positive clones were screened and sequenced.3. The differentially expressed clones in cDNA library were selected and sequenced. All sequences were searched in NCBI database with basic local alignment search tool (BLAST) program. The functional categories of all unique ESTs were performed according to Gene Ontology.Results1. The rats model of TLE:the seizure degrees of rats were judged by Racine grading standard of epileptic seizure. The epileptic rats achieving3-5degree were successful induced. They showed head bobbing, facial muscle twitching, limbs clonus and falling. Two rats of epileptic group were dead and rejected in the experimental group. There was no death in the control group.2. Fourteen different known sequences were revealed after similarity searching by comparing the sequencing results with the data from GenBank (NCBI).3. Systematic analyses of some new genes suggested that cell growth, protein transport, signal transduction, cell-cell adhesion and nervous system development might play roles in epilepsy.ConclusionThe genes associated with epilepsy were identified by SSH method in this research. Therefore, SSH is an effective method for screening differentially expressed genes, and the results of this study are helpful in exploring the molecular mechanism of epilepsy. Purpose:Epilepsy is a chronic recurrent brain dysfunction syndrome caused by abnormal neuron discharge, and is one of the common nervous system diseases.Compared to conventional methodologies, suppression subtractive hybridization (SSH) is a powerful technique to identify rare messages. Moreover, SSH is also a quick method characterized with low incidence of false-positives, sensitive, efficient and good reproducibility.In an effort to understand the molecule mechanism and characterize genes specifically regulated in epilepsy, SSH has been applied in this experiment in which peripheral blood of patients identified by clinical characterization and EEG.Methods1. Extraction the peripheral blood (3ml) of epileptic patients who were identified by clinical characterization and EEG at Shandong University Second Hospital from September until December2011, and store at4℃.2. Total RNA was obtained from peripheral blood. SSH was used for isolating the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pMD-19vector to set up subtractive library which was amplified through E.coli transformation, then positive clones were screened and sequenced.4. The differentially expressed clones in cDNA library were selected and sequenced. All sequences were searched in NCBI database with basic local alignment search tool (BLAST) program. The functional categories of all unique ESTs were performed according to Gene Ontology.Results1. Four different known sequences were respectively from peripheral blood after similarity searching by comparing the sequencing results with the data from GenBank (NCBI).2. Systematic analyses of some new genes suggested that cell growth, protein transport, signal transduction, cell-cell adhesion and nervous system development might play roles in epilepsy.ConclusionThe genes associated with epilepsy were identified by SSH method in this research. Therefore, SSH is an effective method for screening differentially expressed genes, and the results of this study are helpful in exploring the molecular mechanism of epilepsy. Background and purposeThe purpose of this study was to identify new genes that are expressed in the hippocampus of the epileptic rats to increase our understanding about molecular mechanisms underlying the progression of epilepsy.MethodTotal RNA was obtained from hippocampus of epileptic rats and normal rats. Suppression subtractive hybridization (SSH) was used for isolating the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pMD-19vector to set up subtractive library which was amplified through E.coli transformation, then positive clones were screened and sequenced. The validation of the results was carried out by semi-quantitative RT-PCR of the selected genes of interests, i.e. prkc, Trpm6and Ttll11.ResultThere were564white clones and44blue clones in the subtractive cDNA library. Fourteen different known sequences were revealed after similarity searching by comparing the sequencing results with the data from GenBank (NCBI).ConclusionThe genes associated with temporal lobe epilepsy (TLE) were identified by suppression subtractive hybridization (SSH) method in this research. Systematic analyses of some new genes such as spindling-1-like, Acvr2a, TRPM6, and Ttl11suggested that cell growth, protein transport and signal transduction might play roles in TLE. Therefore, SSH is an effective method for screening differentially expressed genes, and the results of this study are helpful in exploring the mechanism of TLE. |
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