Screen The Specific Marked Protein And Approach The Mechanism In The Temporal Lobe Of Drug-Resistance Epilepsy On The Base Of CDNA Library

PART ONE: SCREEN THE DIFFERENTIAL GENE EXPRESSION IN THE TEMPORAL NEOCORTEX OF PATIENTS WITH INTRACTABLE EPILEPSYObjectives: In search for the molecular basis of drug-resistance epilepsy(DRE), we applied a genomic-transcriptomic approach and bioinformatics to identify genes involved in the pathogenesis of DRE. Understanding epileptogenesis, the process by which a normal brain becomes epileptic, may help to establish differential gene cDNA library and find out the new epilepsy associated genes, and then identify the molecular targets for drugs which could prevent epilepsy.Methods: Using a cDNA microarray representing 4096 human genes, we identified that those genes were altered in the temporal neocortex of the patients with DRE and control tissues. Standard stains included H&E,nissl staining and modified bielschowsky silver stains. Total RNA was extracted with UNIzol reagent following the manufacturer's protocol and methods. Scans were performed with the ScanArray4000. The expression value for each gene was then calculated using ImaGene3.0 software (BioDiscovery,Inc). Each experiment was duplicated so that the mean of two ratios for each gene could be used for further analysis. To validate these results, we performed real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) on optional thirteen differentially expressed genes, including MAP1A(microtubular-associated protein1A), MAP1B(microtubular- associated protein 1B),MAP2(microtubular associated protein 2),WDR3 (WD repeat domain 3),CDK5 (cyclin-dependent kinase 5),MAPK14 (mitogen-activated protein kinase 14),GSK-3β(glycogen synthase kinase 3 beta), HSPBAP1 (HSPB associated protein 1), REDD1 (regulated in development and DNA damage responses 1), EML5 (echinoderm microtubule associated protein like 5), MARK1 (microtubule affinity-regulating kinase 1), TRAP220 (thyroid hormone receptor- associated protein), CDR2 (cerebellar degeneration-related protein2)). And we also tested 12 gene by reverse transcription PCR (RT-PCR) (CSNK2A1(casein kinase 2, alpha 1),LAMB1(lamin beta1), MYO1E(myoglobin 1E), TUBD1(tublin deta1), TUBG1(tublin gama1), Cadherin 18(type2), MAP1A,MAP1B,MAP2, CDK5,MAPK14,GSK-3β).Results: 1. Nissl-, H&E and silver- stained neurons showed that the number was significantly decreased in DRE. Patients had cortical neuronal loss and gliosis. Silver-stains showed that there were sprouting of the axon and dendrite and no senile plaques and neurofibrillary tangle formation in the neocortex of patients with DRE; 2. 142 (3.47 %) of 4096 genes significantly altered in the DRE brains. Among the 142 genes, 38(26.8%)genes were down-regulated while 104(73.2%)genes were up-regulated (>2-fold change over controls). These differentially genes included in immune, cell signal transmission, apoptosis, stress-associated, synapse plasticity, structural and cellular reorganization, and so on. Some genes had been reported in previous studies while others had never been reported. 3. Twelve of thirteen by FQ-PCR were coincidence well with the results of microarray scanning. Within the same population, Most of them were found to be significantly increased in the temporal neocortex of DRE comparing to those in the control tissue. 12 genes by RT-PCR were coincidence well with the results of genechip.Conclusions: 1 Multiple genes were associated with DRE, but not a single gene;2 These differentially genes mainly related with immune, cell signal transmission, apoptosis, stress-associated, synapse plasticity, structural and cellular reorganization, and so on; 3 Some genes had never been reported in previous study. It was still unknown to the mechanism of DRE;4 Gene chip had the merits of high-flux and high sensitivity. However, there are some false positive. It was important to test the results by traditional molecular biological technique. PART TWO: SCREEN THE SPECIFIC MARKED PROTEIN AND APPROACH THE MECHANISM IN THE TEMPORAL LOBE OF DRUG-RESISTANCE EPILEPSY ON THE BASE OF CDNA LIBRARYPurpose: The protein is the gene function unit. Make use of the temporal lobe of DRE patients to detect the gene expression of candidate genes. We analyzed the relation of gene and the protein expression and clarify the possible mechanism of DRE.Methods:Used western blot to evaluate and level of extracellular signal-regulated kinase 1(ERK1 ) , ERK2, phosphorylated- ERK1/2(P-ERK1/2),tau protein,phosphorylated-tau protein(ser202, ser404), GSK-3β, phosphorylated-GSK -3β(ser9),MAP2,T cell death- associated gene 51(TDAG51), Insulin-like growth factor-I(IGF-I),CDR2 in 40 surgically resected temporal neocortex of patients with DRE and 20 normal control. Immunohistochemistry (IHC) and immunofluorescence was also used to explore the protein expression and location in DRE.Restuls:As compared with the control group, there appeared significant increased protein expressions of IGF-1,TDAG51,MAPK14, P-MAPK14 (Thr180/Tyr182), MAP2,P-TAU(ser202,ser404),CDK5,GSK-3β, significant decreased expressions of P-GSK-3β(ser9). ERK1,ERK2,and p-ERK in DRE was significant higher than those of controls. They were mainly expressed in the cytoplasm of neuron and glial cell. At the same time, p-ERK expression in the gliosis of DRE was higher than that in the non-gliosis. Western blot analysis showed the results of protein were coincidence with was found in DRE when compared with the normal control brain tissues by immunohistochemistry and immunofluorescence.Conclusion:1, Our work showed that differential mRNA and protein levels may be involved in the pathophysiology of DRE and may be associated with impairment of the brain caused by frequent seizures; 2 , DRE was not caused by a single gene, but many differential genes and proteins,such as TDAG51 associated protein(IGF-1,CDR2,MAPK14, etc), tau associated protein(MAP1A,MAP1B,MAP2,CDK5,etc); 3, these differential genes and proteins expression suggested that DRE have new onset pathway and provide the theory basis of new AEDs.