| Objective Microglia(MG)is a kind of neuroglia with shape of amebiform and function of phagocytosis and broadly distributed in the central nervous system.Microglia originated from mononucleocyte in the circulation system and functioned as the supporting system for the neuron,serves as the main immunological system in the central nervous system.Microglia is in quiescent status when central nervous system is not damaged,but when central nervous system were damaged,the microglia would be activated and would possess the ability of a specific phagocytosis in that way the apoptotic neuron and other invaded matter would be eaten.When the damage was repaired or cured,the microglia would return to the quiescent status and at this situation it does not have the ability of phagocytosis.Epileptic status is a disorder of brain temporary dysfunction caused by the sudden abnormal discharge of the neuron in the brain.In the epileptic status,the membrane potentials of the neuron was transferred from the quiescent into action potential and stimulate the neuron to release a large amount of neurotransmitters from the presynaptic membrane and which would bind to the relevant receptor in the postsynaptic membrane and keep the neuron to enter a persistent excitatory state and accelerate the apoptosis of the neuron.At the mean time of the epileptic onset,the microglia would be activated and play a role of phagocytosis in this way to timely clean up the abnormal dead neuron and the poisonous substances produced in the epileptic onset so that to play an important role in the maintenance the relative stability of the body internal environment.The number of the activated microglia increases and the cell body are large,and the processes increase also.To investigate the level of activation of microglia,the degree of the damage of the neuron induced by epilepsy constant onset would be indirectly reflected.The degree of the activation of the microglia is different according to the variant of the timing range of the status of epileptic.Purαis a wildly existed protein in the body which has high affinity to bind to the single stranded DNA and RNA,it has regulatory effects on DNA replication,transcription,and RNA transport and protein translation.Furthermore,Purαalso participates in the process of repairing the damaged DNA.The neuronal cytoplasm has abundant amount of Purα.The recent studies have demonstrated that Purαplays an important role in the neuronal development and the expression of Purαis also closely associated with some neurodegenerative diseases.The aim of the current study is focused on the effects of Purαon the expression of P2Y12 receptor in the BV2 cells,and the relationship between the microglia activation and the expression of P2Y12receptor at the status of epileptus as well as the effects of Purαon the expression of P2Y12 receptor.Methods1.To establish the model of rat epilepsy.LiCl-Pilocarpine was used to establish the rat epileptic model.The advantage of the method is classic and with higher efficiency of success.2.Packaging lentivirus of Pur overexpression viron and Purαknock down viron:Lentivirus were packaged with lentivirus packaging plasmid pCMV-DR8.2 dvpr and pCMV-VSV-G according to the instruction of Gene hem biological Co.the lentivirus package was successful and the packaged viron has high titers and meet the requirement of the experiment.3.Injection of lentiviron into the animal brain:Guiding with stereotaxic apparatus,20μl of Purαknock down lentivirus pLKO.1-Purαand overexpression lentivirus pCDH-EF-1-Purαwere injected into the CA1 region of rat hippocampus separately of Purαknock down and overexpression groups with micro syringe.15 days after the microinjection,the animals were killed and the brain tissue slice was prepared to check the Purαprotein expression status was checked in the brain slice under inflorescent microscope.4.Immunohistochemistry:(1)The tissue samples from different time point in epilepsy and control groups were collected and the status of microglial activation in hippocampus region was assessed with immunohistochemistry method.(2)The rat brain tissues of persistent epileptic animals in 2 hours’time point were collected from lentivirus group,control group and N.S group and the status of microglial activation were evaluated with immunohistochemically method.5.Western blotting:(1)The cellular total proteins from BV2 cells in different groups(Control group,transfected with pCDNA3.0 empty plasmid,overexpression group,transfected with pCDNA3-Purαand knock down group,transfected with pLKO.1 shRNA-Purα)were extracted and about 60μg total cellular proteins were applied to SDS PAGE to check the status of P2Y12receptor protein expression.(2)The animal brain tissue proteins from the different time point of the persistent epilepsy and the normal control groups were extracted and about 50μg brain tissue proteins were applied to the SDS-PAGE to the check the status of protein expression of P2Y12 receptor,CREB and Purα.(3)The brain tissue protein of persistent epileptic rat for 2hours in hippocampus from lentivirus group and normal control group were extracted and about 50μg proteins were applied for SDS-PAGE to evaluate the level of protein expression of P2y12 receptor,CREB and Purα.Results1.The rat epileptic model induced with LiCl-Pilocarpine was successfully established and degree of epileptic onset matched the requirement of the experiment.2.Purαwas successfully expressed in the rat hippocampus tissues in the 15~thh day after the viron injection and which was confirmed with the examination of GFP under the inflorescent microscope.3.Microglia were activated at the status of epilepsy,in the different time point of the persistent epileptic onset,the level of microglial activation was variant,the microglial activation reach its peak at the 2 hours’time point of the epilepsy.Compared with normal control group,the number of activated microglia,the volume of activated microglia and the number of the processes and the branches of the processes of the activated microglial were all at their peak in the 2h epilepsy group.4.Western blotting:(1)The expression levels of P2Y12 receptor protein were higher in Purαoverexpression groups of BV2 cells and rat hippocampus tissues of the epileptic group than that in the empty control groups.(2)In the different set time points,0h,1h,2h,3h,4h,of the epileptic onset,the protein expression levels of P2Y12 receptor protein and Purαwere at the highest status than other time points;the expression level of CREB were at the peak in the 1 h time point of the epileptic status.Conclusion1.The continuous status of epilepsy induced the activation of microglia,the degree of microglial activation is related to the time point of the persistent epilepsy.2.The protein expression levels of P2Y12 receptor,Purαand CREB were variant in the different time point of the epilepsy.3.Purαcould up-regulate the gene expression of P2Y12 receptor. |
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