| There are many possible mechanisms of ischemia cerebral vascular disease including the release of excitatory amino acids mainly Glutamte. calcium influx and overload as well as the toxicity of NO. It was reported that Glutamate plays a key role in the process of neuronal ischemia and hypoxia injury. But it is not the single mechanism of it.BDNF and GDNF are known to have important functions in neuronal survival, differentiation and plasticity, in addition to their roles as survival-promoting factors, BDNF and GDNF reportedly can enhance neuronal cell injury in some cases, for example, the death caused by excitotoxicity, glucose or oxygen deprivation. The celluar mechanism of the death enhancing effect of BDNF and GDNF remain unknown, in contrast to that of its survival-promoting effect, especially the relative of apoptosis and the BDNF and GDNF.In view of the above mentioned problems, the following experiments were designed: first of all, two hypoxia models were made in cultured hippocampalneurons by the addition of Glutamate or by the deprivation of oxygen. Thecell survival rate and cellular morphological changes were used to indicatethe degree of celluar injury. Secondly, the adenoviral vector was used totransfer the gene of BDNF, GDNF into the hippocampal neurons in vitro.Thirdly, in the two models in vitro we mainly examined the protective effectof Ad-BDNF and AdGDNF on the apoptosis, which has been observed usingthe method of in situ terminal transferase mediated dUTP-biotin nick endlabeling(TUNEL). At the same time, the bcl-2 protein expression inhippocampal neurons was observed by the method of immunohistochemistry.The following experimental results were obtained by the study:I.We have successfully established the hippocampal neuronal cell culture andobserved cell growth and maturation in a period of month. The cell growthwas the most vigorous in 7d-lOd, during which period the neurons werevulnerable to hypoxia injury. Immunohistochemically, the MAP2 positiveneurons comprised more than 90% of the total cells.2.Through depriving the supply of oxygen or exogenous addition of Glutamate, we established cultured hippocampal neurons hypoxia model and Glutamate excitotoxicity model successfully. It is considered that hippocampal cells in these two models might be a suitable object for the research of hippocampal neurons injury due to hypoxia and Glutamate excitotoxicity and screening out protective agents of neurons. 3.Our results show that hippocampal neurons could be infected successfullyby adenovirus, and the infective rate could reach 30%.4.The Ad-BDNF and Ad-GDNF could decrease the apoptotic rates of hippocampal neurons in Glutamate and hypoxia models, at the same timeboth of them could induce the expression of bcl-2 of the neurons suffering the two kinds of insults in vitro. It was observed that the protective effects of BDNF and GDNF were depending on the expression of BDNF and GDNF transferred by the adenovirus.5.Bcl-2 protein in hippocampal neurons has structural expression in normal condition. The synthesis of bcl-2 protein lowered after hypoxia and Glutamate insult. Both of Ad-BDNF and AdGDNF could hold back the down-regulation of bcl-2 protein in different degree. The protective effects of Ad-BDNF and AdGDNF on the apoptosis may probably related with the increase of the expression of bcl-2 protein.AbbreviationGlu: GlutamateBcl-2: B-cell lymphom/IeukemiaBDNF: Brain-derived neurotrophic factorGDNF: Glial cell line-derived neurotrophic factorMAP2: Microtubule-association protein 2NTF: NeurotrophinCas: Caspase... |
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