Protective Effection Of Fas SiRNA On Neural Cells After Ischemia-reperfusion Injure

Main goal of cerebral vascular disease treatment is to save the cells aroundthe ischemic core . Ischemia-reperfusion insult plays an important role in thepathology change and apopotosis is an essential machnism . apopotosis dependson some gene expression which consist of apoptpsis and antiapoptosis gene .Fas is the most important one of them.RNAi is a new method which is now used in gene function research andgene treatment.It can specificely inhibite a gene expression at the posttranscription level.Theorylly Fas siRNA can inhibite Fas , caspase-3geneexpress, downregulate caspase-3 activty and apoptosis rate.objectsTo establish an neuron ischemia-reperfusion insult model on NGF treatedPC12 cell ,study the change in Fas mRNA ,Fas protein and caspase-3 proteinexpression level and inhibition effect of Fas-RNAi on neuron apoptosis afterischemia-reperfusion.Experiment one Methods PC12 cells induced in to neuron by NGF weredivided into 6 groups: control group,which was hatched in normal culture;ischemic 30min,60min,90min,2h,6h groups,which respectively underwent30min,60min,90min,2h,6h ischemic treatment with balanced salt in 5%CO2,95% N2,37℃。After the ischemic treatment,cells were hatched in normalculture,thus to stimulate ischemia-reperfusion insult on neural cells. At 0h ,1h ,3 h ,6 h ,12 h ,24 h , 72 h time point after reperfusion,cell activity andapoptosis rate were tested.Result In the control group,apoptosis and cell deathhappened during the cell culture,but the rates were very low,the apoptosis ratewas no more than 8.2% and death rate 1.8%. In ischemic 30min , 60mingroup, apoptosis rate became obviously higner after reperfused 12 h and reachedpeak at 24h,apoptosis rate reached 20.7%.The death rate increased 5.8% at12h.In ischemic 90min group, apoptosis rate became obviously higner afterreperfused 6h (20.9 %,P<0.05) and reached peak at 12h(28.7%,P<0.01).The death rate was 9.7%at 12h(P<0.01).In ischemic 2h , 6h group,apoptosis rate was much more than the other groups ,reached 32.7%(P<0.05)at 6h,and at12h reached its peak 35.3%,but the death rate increased to 28.6%。The 90min group had a higher apoptosis rate and a early peak compared with30min,60min group, and a higher apoptosis/death rate. Conclusion The NGFtreated PC12 cell can well mimic neuron ischemia-reperfusion insultmodel.choose the mimic 90 min ischemia before reperfusion as model.Experiment two Methods Cells were divided into 2 groups: controlgroup,which was hatched in normal culture and model group on whichischemia-reperfusion insult model were made:the cell underwent 90minischemic treatment and then insulted with reperfusion.At 0h,1h,3h,6h, 12h, 24h,72h time point after reperfusion,cell activity , apoptosis rate,DNA ladder andfas,caspsae-3 exppression level were tested.ResultIn control group,change of the cell activity and apoptosis rate between eachtime point were unobvious.In model group,after reperfusion,cell activity becamedecreased and the change became obviously at 3h time point (P<0.05),at 24hreached its lowest level(P<0.01).Apoptosis rate of cells in model group washigher than those in the control, the difference became obviously at 6h timepoint and reached its peak at 24h. Typical DNA ladder was seen in modelgroup at 12h time point. Fas positive rate was obviously higher in medol groupat 3h and reached its peak at 6h. Caspase-3 positive rate was obviously higher inmedol group at 6h and reached its peak at 24h. Conclusion Fas and caspase-3expression level increased after ischemia-reperfusion insult.Experiment three Methods PC12 Cells treated with NGF weredivided into 5 groups:control group which was hatched in normal culture andmodel group on which ischemia-reperfusion insult model were made, vectorgroup which was transfected with vector 24 hours before the models were set up.siRNA 2 group and siRNA 1 group which were respectively transfected withpSIFas-A and pSIFas-B 24 hours before the models were set up. Fas mRNAand protein exppression level were measured at 6h time point with RT-PCR andWesternblot respectively .Result In siRNA 2 group,Fas mRNA and proteinexpression became obviously lower than that of the model,vector and siRNA 1groups at 6 h time point,and was not obviously different from the controlgroup,which suggest that the siRNA successfully inhibite the Fas geneexpression. Conclusion Fas-siRNA was successfully contrasted and inhibite thefas gene exppress .Experiment four Methods Cells were divided into 5 groups:control groupwhich was hatched in normal culture and model group on whichischemia-reperfusion insult model were made, vector group which wastransfected with vector 24 hours before the models were set up. siRNA 2 groupand siRNA 1 group which were respectively transfected with pSIFas-A andpSIFas-A 24 hours before the models were set up. At 24h time point afterreperfusion,cell activity , apoptosis rate were tested by FCM and TUNNELproccesure. Caspase-3 activity were measured at at 24h time point.Result InsiRNA 2 group,caspase-3 activity became obviously lower than the model,vector and siRNA 1 group at 24h time point,and was not obviously differentfrom the control group. In siRNA 2 group,apoptosis rate became obviouslylower than that of the model,vector and siRNA 1 groups at 24h time point,andwas not obviously different from that of the control group. ConclusionFas-siRNA downregulated caspase-3 activity and cell apoptosis rate.ConclusionThe NGF treated PC12 cell can well mimic neuron ischemia-reperfusioninsult model.choose the mimic 90 min ischemia before reperfusion as model.Fas and caspase-3 expression level increase after ischemia-reperfusioninsult.Fas-RNAi transfection can specially suppress expression of Fas gene andprotect neural cells from apopotosis after ischemia-reperfusion injure.