The Experimental Study Of Construction Of Eukaryotic Cell Expression Plasmid For HIGF-1 And Its Effect On Apoptosis Of C6 Cell In Vitro And Neural Cells Of SCI Rats In Vivo

It is a worldwide subject for therapy of spinal cord injury (SCI), genetherapy is an ideal method to prevent and treat human disease in the future.Although there have been taken amazing outcome on gene therapy of SCI inexperiment study, for the complex effect factor and injury mechanism, there isstill a lot of key point unknown for people to study incontinuous. Series of study of effect of insulin like growth factor (IGF-1) on neuralsystem have been published in recent years, but the study always make its focuson neual cell culture in vitro and traumatic brain injury, No report have beenpublished about effect of IGF-1 on acute spinal cord injury in vivo. So we workout and do the study to invest the intervene effect of IGF-1 on apoptosis phaseof SCI rats.spinal cord neural cells. For the discovery of NGFs moleculecharacter and secondary injury in central nervous system, gene therapytechnique, in some time, is promising method that have possible to cure SCI,nowm there re a few experimental report published in recent years. The directly injection method could be use to transplant neural growthfactors(NGFs) into spinal cord after SCI, but a tube should be placed in a longterm for the NGFs,giant molecule, is hard to permeation through blood-brainbarrier.genetically modified cell maybe a method to substitute. Takahashi et alutilize recombine Bcl-2 plasmid to treat SCI rats, result show that obviouslyexpression of IGF-1 gene in the injury zone and somewhere 1.0cm cranial orcaudal to focus, and the expression last to at least 7 days (no more than 14days).In the study, recombinant IGF-1 plasmid was constructed and used to interveneSCI rats.In the study, human IGF-1 DNA was choosed as therapy gene, recombinanteukaryotic expression plasmid, pcDNA-IGF-1, was constructed ex vivo.Fragment electrophoresis and gene sequence measurement were taken to ensurethe inserted gene;The recombinant plsmid was transfected into neuroglioma cell(C6 cell) with liposome-mediated method and positive transfected cell strainwas selected by G418. immunohistochemistry , flow cytometry(FCM) weremeasured to ensure gene expression. C6 cell apoptosis was Induced in vitro,FCM result show, IGF-1 could reduce C6 cell apoptosis in vitro.Semi-transsection SCI model was made in vivo, two research staff who attendthe experiment and master the BBB score system observed and evaluate SCIrats' behaviour., average score of two people was used at last. The BBB score isrised with comply with time, compared with model group, treatment group has amore sharp rise tendency in 1 week postoperation. Between 2-4 weekspostoperation, the BBB score are both rised slowly in two groups. Totally, theBBB score is higer in treatment than that of model group. Transmission electronmicroscope (TEM) was taken to observe SCI rat spinal cord, the result show, inmodel group, lot of neurone apoptosis, in IGF-1 treatment group, quantity of theapoptosis cell is reduced significantly. TUNEL result show, there are a littleapoptosis cells (2.2±0.7) in blank group, in model group, apoptosis cells is muchmore than that of blank group, in treatment group, cell apoptosis is suppressedsignificantly 1 or 2 weeks postoperation. The difference of treatment groups isinsignificant.It is significant for reduction of neural cells apoptosis and secondary injuryto save neural cells and promote neural regeneration. In SCI, apoptosis cellshave time distribution character, in acute phase, it will be different result fortherapy in different time. Time distribution character are that the cell apoptosisis begin in 24h postoperation;in 72h postoperation, the cell apoptosis go toclimax in neibour segment and the quantity is more than that of focus;apoptosisphenomenon would last to 21days postoperation;glial cell apoptosis would lastto long time and neuron apoptosis is happen in early time.so there maybe havetherapy window. Lee found in his study about bFGF therapeutic act on SCI, It sno obviously act for transfection delay to 3hs postoperation. In the study,pcDNA-IGF-1 was used to intervene SCI rats immediately postoperation, andobserve exogeneous expression and neural cell apoptosis 1day, 3days, 7days,14days, 21days and 28days postoperation.Experimental result shows that transfected gene plasmid mediated bylipofectamine is excellent vector for gene therapy for SCI. it is asepsis,disimmune and inertia;it could be biodegradation and easy to produce andutilize;it coule be transfect neural cell orientable;it have high transfect rate andcould be enough for demand of gene therapy.Experimental result in vivo shows that it is feasible for pcDNA-IGF-1transfection in vivo mediated by lipofectamine. Expression of IGF-1 isaccommodate to the apoptosis of neural cell, recombinant plasmid would bedegradation in 2 weeks postoperation gradually, and exogenous IGF-1expression could last to 3 weeks postoperation and reduce neuronal apoptosissignificantly.The study investigate the feasibility of gene therapy for SCI byrecombinant IGF-1 plasmid, and effect of IGF-1 on neural cell apoptosis in SCI.the result is important for research the other effect factor of neural cell apoptosisin SCI, therapeutic mechanism of IGF-1 in SCI and clinical application of genetherapy in SCI further.