Expression Of Proteins And Genes Involved In Growth Cone In Intractable Epilepsy

PART ONE:EXPRESSIONS OF GROWTH CONE RELATED GENES IN THE TEMPORAL LOBE BRAIN TISSUE OF DRUG-RESISTANT EPILEPTIC PATIENTS DETECTED BY GENE CHIPObjectives: To investigate the expressions of growth cone related genes in the temporal lobe brain tissue of drug-resistant epileptic patients and explore its effects on the formation of susceptibility and the abnormal growth of axon.Methods: Both RNAs extracted from the temporal lobe brain tissue of the 40 cases of drug-resistant epileptic patients and 14 cases of control group were reversely transcribed to the cDNA incorporated with the fluorescent molecule to act as a probe; PCR products of 4096 human genes were spotted onto a chip in array and after chip hybridization and strict film processing, the images of fluorescent signals in the array were scanned by a ScanArray4000 fluorescent scanner and its results were analyzed by GenePix Pro 3.0 soft ware and then the expressions of growth cone related genes in the temporal lobe brain tissue of drug-resistant epileptic patients and health controls were analyzed by computer.Results: The genes such as integrinα2 mRNA, lamininβ1 mRNA,coronin1CmRNA , myosin1EmRNA , tubulin gama1mRNA , neural adhesion molecule NB-3mRNA,neural cell adhesion molecule2mRNA, cadherin18mRNA, sorting nexin 17 mRNA , microtubule-associated protein2mRNA, microtubule-associated protein 1AmRNA were up-regulated , and those such as tubulin deta1mRNA, microtubule-associated protein1BmRNA were down-regulated in the brain tissue of drug-resistant epileptic patients.Conclusion: An expression abnormality of growth cone related genes detected in the temporal lobe brain tissues of drug -resistant epileptic patients, growth cone may be associated with impairment of the brain caused by frequent seizures , suggested that the recurrent attacks of epilepsy can influence the formation of the susceptibility and the abnormal growth of axon by influencing the abnormal growth of the growth cone. PART TWO:EXPRESSION OF LAMININβ1 AND INTEGRINα2 IN BRAIN TISSUE OF PATIENTS WITH INTRACTABLE EPILEPSYObjective To investigate the expression of lamininβ1 and integrinα2 in the brain tissue of patients with intractable epilepsy and explore its role in the pathogenesis of intractable epilepsy.Methods FQ-PCR , immunohistochemistry , immunofluorescence and western blotting were used to test the expression of lamininβ1 and integrinα2 in the surgically removed brain tissues of patients with intractable epilepsy from the brain bank of our department (n=40, temporal lobe=32, hippocampus=8) ,and the results were compared with those of controls.Results FQ-PCR confirmed that the expression of lamininβ1 and integrinα2 in the brains tissue of patients with intractable epilepsy increased, the relative copy numbers of temporal lobe were more than the controls(lamininβ1mRNA : 8.09E-03/4.81E-03 ; integrinα2mRNA :3.08E-01/1.49E-01,P <0.05), the relative copy numbers of hippocampus were more than temporal lobe in intractable epilepsy group (lamininβ1mRNA : 1.10E-02/6.80E-03 ; integrinα2mRNA :5.95E-01/2.50E-01,P <0.05). Lamininβ1 and integrinα2 protein expression were significantly increased in temporal lobe cortexand and hippocampus as compared with controls in the same regions (immunohistochemistry optical density : lamininβ1:0.3597±0.0108/0.0967±0.0269 in temporal lobe tissue, 0.5431±0.0129/0.0715±0.0203 in hippocampus tissue;integrinα2: 0.4171±0.0219/0.0374±0.0094 in temporal lobe tissue , 0.5381±0.0194/0.0526±0.0103 in hippocampus tissue, P <0.05). Lamininβ1 and integrinα2 immunofluorescence stain accumulated in cell membrane and cytoplasm, showed strong fluorescence intensity in the brains tissue of patients with intractable epilepsy. Lamininβ1 and integrinα2 protein expression were significantly increased in temporal lobe cortexand and hippocampus as compared with controls in the same regions by western blotting(OD: lamininβ1, 0.6508±0.1795/0.3374±0.0937 in temporal lobe tissue , 0.7503±0.1975/0.3431±0.0498 in hippocampus ; integrinα2, 0.5343±0.0694/0.3095±0.0526 in temporal lobe tissue , 0.9043±0.0921/0.5951±0.0827 in hippocampus,P <0.05).Conclusion This work showed that an increasion in lamininβ1 and integrinα2 may be associated with impairment of the brain caused by frequent seizures, which may play an important role in the pathogenesis of human intractable epilepsy.We suggested that the recurrent attacks of epilepsy can influence the formation of the susceptibility and the abnormal growth of axon by influencing the abnormal growth of the growth cone and this may be the reason causing the formation of drug resistance in epilepsy.