| Objective This study adopts the primitary culturet rats hippocampal neurons in vitro to simulated cerebral ischemia hypoxia model,with the combination of main active ingredient in astragalus and ligusticum chuanxiong hort:astrugaloside iv、lingustruzine、ferulic acid、ligustilide as the study object,to investigated the protective effect of that on tats hippocampasl neurons injured by oxygen and glucose deprivation from the cellular level and molecular level,offer the clinical treatment of ischemic cerebrovascular disease with clinical thinking and further reference.Methods 1.Cultured primary rats hippocampal neurons and identification:taking rats born within 24 hours as material,separation hippocampus of both side,digesting it with trypsin,centrifugal,sieving to suspension,vaccinating it in the cell culture plate,.After celsl growing into matured,conducting immunological identify by neuron specificity enolization enzymes(NSE).2.Taking MTT toxicity assay to determine each non toxic dose of main active ingredient in astragalus and ligusticum chuanxiong hort:astrugaloside iv、lingustruzine、ferulic acid、ligustilide Selecting appropriate nose to group:normol group,oxygen and glucose deprivation model group,nimodipine group,and active ingredient of astragalus and ligusticum chuanxiong hort compatibility group3.Establishing the hippocampal neurons injured model:Taking matured hippocampal neurons as material,abandon the original culture medium,reentering OGD fluid with corresponding drugs,then placing them into anoxic box,ventilating it with missed gas((1%02+5%CO2+ 5%N2)),puts it into 37℃ and 5%CO2 incubator continue to culture four hours.4.Observing the oxidative damage effect of ingredient compatibility group on hippocampal neurons injured by oxygen and glucose deprivation,detect the LDH leakage content in supernatant、the malondialdehyde level,super-oxide dismatase activity,glutathione preoxidase(GSH-PX)activity and NO content of supernatant.5.Observing the apotosis effect of ingredient compatibility group on hippocampal neurons injured by oxygen and glucose deprivation:the apotosis rate of hippocampal neurons was measured by using Hoechst33342 nuclear staining and the flow cylometric assay.The activity of caspase-3 also detected to asses the apotosis rate of cells.Results 1.Primary hippocampal neurons morphology and result of identificate:After cultured 7 days,the primary hippocampal neurons body become bigger,has high light quality and holo,the neurons dendrites become longer and wilder,the cells network interweaved.The neuron-specific enolase nuclear staining result is that cytoplasm and some relatively bulky protrusome were dyed tan or brown and nuclears was dyed pale people.The rate of positive cells is more than 90%.It turns out that this method cultured high purity hippocampal neurons.2.The effect on shape of hippocampal cell injured by oxygen and glucose deprivation:After cells suffer from oxygen and glucose deprivation for 4 hours,the hippocampal neurons body swelling,its refractin is reduced and protuberant is fructyred.Cell nucleus pycnosis and rupture occurs in the body,thenetwork between cells are destroyed.3.Toxicity experiment research:The MTT assay determine the safe dosage of each effective component of astragalus and ligusticum chuanxiong hort.The best dosage of them respectively were:Nimodipine best dosage is.Each effective component concentration orthogronal compactly 9 groups.Conclusion 1.In this research,we successfully cultured the typical hippocampal neyrons and established hippocampal neurons oxygen and glucose deprivation model.2..The astragalus and ligusticum chuanxiong hort effective components compatiboloy have obvious protective effect on hippocampal neurons injured by OGD.3.The mechanism of protective effect maybe:It can strongly protect hippocampal neurons complete structure and shape、enhance the ability of clearning oxygen free radicals、reducethe activity of caspase-3 and anti apotosis. |
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