Construction Of Retroviral Vector Encoding GFP And The Application Of Retrovirus-mediated Cell Labeling In Hippocampal Neurogenesis In Epileptic Rats

Epilepsy, characterized with transient brain dysfunction caused by theaberrant discharge of neurons, is a chronic and commonly neurological disorder.Recently, many progresses in the research of epileptic mechanism have beenmade and several hypotheses about epileptic mechanism have been proposed.However, the underlying mechanisms of this disorder remain disputable andunclear. Increasing evidences suggest that aberrant neurogenesis associated withseizures not only lead to cognitive dysfunction following epilepsy but also isregard as an underlying mechanism of abnormal neural circuits, hippocampalsclerosis and reconstruction of synapses, and these pathological alternations maybe epileptogenic. Thus, as an important epileptogenic factor, aberrantneurogenesis has been causing increasingly concerns.In our preliminary studies, we found that seizures could direct newborn granular cells migrating to the hilus abnormally, and IL-1β, one of key factorsof inflammatory cascade, could induce the migration of cultured neurons in vitro.So we hypothesize that up-regulation of IL-1βcaused by seizures result inectopic cells in dentate hilus and may be an etiological factor of epilepsy.Retroviral vector is a commonly used tool in molecular biology research, andthe morphological structure of newborn cells can be observed clearly byretrovirus-mediated cell labeling. As a result, we have designed and fulfilled thisstudy, and hope to throw light on further understanding of the pathogenesis ofepilepsy.Objective:This thesis devoted to construct a retroviral vector encoding GFP and useit to analyze the impact of epileptic activity on adult-generated cells in thedentate gyrus in vivo, detect expressions of IL-1βand its mRNA, and constructa recombinant retroviral vector for RNA interference targeting the IL-1R1gene.Methods:1、With the plasmids MSCV-IRES-GFP and pMCV-Ecopac, we packagedretroviral viruses expressing GFP in HEK293T cells for subsequent studies.2、 One week after lithium-pilocarione-induced seizures, stereotacticinjections of2ul retrovirus were placed into both sides of dentate hilus of allexperimental rats. Western blot and Real-time PCR were used to measureexpressions of IL-1and its mRNA. The migration of newborn cells was detectedby immunofluorescent method.3、We cloned siRNA of IL-1R1into MSCV-IRES-GFP, and then obtainedcorresponding retroviral viruses. Results：1、It was confirmed that about80-90%of HEK293T cells infected with theconcentrated retroviral viruses can express GFP after48h.2、After seizures, expressions of IL-1and its mRNA were increasedsignificantly in dentate hilus, and proliferation was enhanced and newborn cellsmigrated into hilus abnormally in experiment group. Ectopic cells and aberrantdendrites could be induced by epilepsy.3、The construct expressing siRNA of IL-1R1was confirmed by sequenceanalysis before being packaged into the retroviral viruses. It showed that90%ofHEK293T cells were successfully infected with the corresponding viruses andexpressed reduced amounts of IL-1R1mRNA compared with the control cells.Conclusion：1、Newborn neurons labeled by retrovirus migrate into the dentate hilusfrom SGZ abnormally after seizures in vivo, and become ectopic cells whichmay give rise to epileptogenesis. Two sorts of retrovirus have been constructedsuccessfully.2、Expressions of IL-1βand its mRNA were up-regulated after epilepticseizures. The recombinant retrovirus for the RNA interference of IL-1R1genewas also constructed successfully. Both of them will lay a foundation forinvestigate the molecular mechanism of the aberrant migration of newborn cellsassociated with seizures.