| Different diseases show free radicals as factors in the pathogenesis and the nervous system is no exception. Cell discharge may cause an increase in free radicals, such as superoxide anion. By inhibiting the effect of glutamate synthetase, free radicals cause an increase in glutamate, an excitatory neurotransmitter, inducing epilepsy. Studies have shown that the increase in free radicals, the radical reactivity enhancement, which will cause a change of the configuration, structure and permeability of the cell membrane. This change also changes the neuronal membrane and synaptic function, density and distribution of membrane ion channels and threshold, which affect Na Ca flow and enter cell. The effect cause of neuron depolarization shift (depolarization shift, DS) and repeated, excessive synchronous discharge that is, causing the clinical seizures. However, organisms also have protective mechanisms, such as inhibition of the oxidation occurs. The antioxidant factors contain superoxide dismutase(SOD), catalase(CAT), glutathione peroxidase(GP), glutathione reductase(GR), peroxidase(POD) and glucose-6-phosphatase(G6P). Many studies have reported the relationship that may exist between SOD and epilepsy.A great many studies have demonstrated that genetic mutation is closely associated with epilepsy, particularly the genetic mutation of sodium channels. There are11sodium channel a-subunit genes among human protein coding genes. Of them, only SCN1A, SCN2A, SCN3A and SCN8A are expressed in the central nervous system, and the sodium current produced by NaV1.1and NaV1.2, i.e. the products of SCN1A and SCN2A, accounts for70%of the total sodium current in the central nervous system. It has been confirmed that the mutational SCN2A is related to epilepsy, sensitivity to pain, and other diseases of nervous system. Recent studies determined that sodium current in the inhibitory intercalary neurons in mice with SCN2A knocked out significantly reduced, while, and there was no significant change in sodium current in the excitatory pyramidal neurons. This caused an increase in excitability of the nerve circuitry. The results provide a good explanation that an decrease in the expression of SCN2A is the cause of epilepsy. Hence, the expression level of SCN2A is of vital importance in maintaining the normal function of the central nervous system.This paper studies the changes of SCN2A and Cu/Zn SOD concentration between in gene expression in the cerebral cortex of temporal lobe epilepsy and normal tissue. At the same time, we explore the SH-SY5Y human neuroblastoma tumor cells carrier of SCN2A interference after transfection as wll as the copper-zinc superoxide dismutase expression and enzyme activity changes, In addition, cell levels interference SCN2A vector and Cu/Zn SOD expression vectors were co-transfected and SCN2A and Cu/Zn SOD treatment effect is described. Chapter one Temporal lobe epilepsy patients with cortical tissue SCN2A and Cu/Zn SOD expression characteristicsObjection:Study temporal lobe epilepsy patients with cortical tissue SCN2A expression and Cu/Zn SOD content and activity.Methods:Using Realtime PCR and western blot analysis the expression of SCN2A temporal lobe epilepsy patients with cortical tissue and normal control group. By employ elisa to detect Cu/Zn SOD content and Cu/Zn SOD enzyme activity. Differential expression was analyzed using statistical methods.Result:Patients with temporal lobe epilepsy cortical tissue SCN2A in the expression level of mRNA expression was significantly lower than that in normal control group, and the difference was significant (p<0.01). Western blot technology, there are seven temporal lobe epilepsy patients with cortical tissue samples can not be detected the SCN2A protein expression by Western blot. Four samples detected SCN2A protein expression, but the expression level than normal control group was significantly lower. Expressed in patients with temporal lobe epilepsy cortical tissue expression was significantly lower than the normal control group, and the difference was highly significant (p<0.001). Expressed in temporal lobe epilepsy patients with cortical tissue expression level was significantly lower than the normal control group, and the difference was highly significant (p<0.001).Conclusion:Expression of SCN2A and Cu/Zn SOD of cortical tissue in patients with temporal lobe epilepsy may have a potential contact, may play a role in patients with temporal lobe epilepsy and co-occurrence of the disease mediated development. Chaptor2SCN2A interference effects on Cu/Zn SOD enzyme activity in SH-SY5Y human neuroblastoma tumor cellsObjection:SH-SY5Y human neuroblastoma tumor cells SCN2A interference, and to explore the impact of interference on the Cu/Zn SOD enzyme amount and enzyme activity.Methods:Build SCN2A interference vector, cells transfected with the expression of of SCN2A genes interfere with SH-SY5Y human neuroblastoma tumor cells. And then using Realtime PCR and western blot analysis the expression of SCN2A interference group and the normal control group. Using the ELISA method to detect the content of Cu/Zn SOD, Cu/Zn SOD activity was assayed by detection of Cu/Zn SOD enzyme activity. And use statistical methods to analyze the differential expression.Result:Realtime PCR results showed that the transfection interfere with the carrier group was significantly higher than that in the control group (P <0.05). Western blot results display SCN2A downregulated expression at the protein level after transfection of the interference vector. Immunofluorescence results also obtained similar results, as the protein SCN2A downregulated expression after SCN2A interference. Cu/Zn SOD content and enzyme activity of SCN2A interference carrier group were significantly lower than the expression level of the empty vector control group, and the difference was highly significant (p<0.001). Conclusion:In suppression SCN2A expression in SH-SY5Y human neuroblastoma tumor cells can cause reduce of content and enzyme activity of Cu/Zn SOD. Chapter3SCN2A interference effect on the protection of Cu/Zn SOD on SH-SY5Y human neuroblastoma tumor cellsObjection:SH-SY5Y human neuroblastoma tumor cells SCN2A interference and Cu/Zn SOD expression, study the Cu/Zn SOD protective effect on SH-SY5Y human neuroblastoma tumor cells after SCN2A interference.Methods:The cells were divided into four groups, including the control group, the empty vector, SCN2A interference group and SCN2A interference vector+Cu/Zn SOD over-expression vector group. Respectively, the vector was transfected into SH-SY5Y human neuroblastoma tumor cells. Realtime PCR and western blot analysis the expression of SCN2A interference group and the normal control group. The content and activity of Cu/Zn SOD were assayed. And use statistical methods to analyze the differential expression.Result:The constructed SCN2A interference vectors were successfully transfected. The highest SCN2A expression was detected in control group and empty vector group, the lower SCN2A expression was detected in group of SCN2A interference vector+Cu/Zn SOD over-expression vector group, and the lowest SCN2A expression was detected in SCN2A interference group. The content and activity of Cu/Zn SOD in control group and empty vector group were the highest, those in group of SCN2A interference group and SCN2A interference vector+Cu/Zn SOD over-expression vector group were lower, and those in SCN2A interference group were the lowest.Conclusion:SCN2A influence the expression of Cu/Zn SOD content and enzyme activity. Cu/Zn SOD have protective effect on SH-SY5Y human neuroblastoma tumor cells after SCN2A interference. Thus these two factors closely associated. |
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