Effects Of SiRNA-smad3 In ActA/Smads Signalling Pathway On Ischemic Brain Injury

Previous research indicated that the proteins of ActA and Smads had been expressed in center nervous system. ActA/Smads signaling pathway has significant roles in some diseases of center nervous system, but the particular mechanism is not distinct. This study paid attention to the ActA/Smads signaling pathway, combined with extracorporal experimentation on neurons transformed from PC12 cells.After precondition of exogenous ActA and oxygen and glucose deprivation (OGD), viability and apoptosis percentage were measured with the method of MTT and Hoechst33342 staining, and mRNA expression of caspase3 and the key sites of ActA/Smads signaling pathway were measured with the method of RT-PCR in order to illustrate the effect of neurons transformed from PC12 cells subjected to OGD and ActA/Smads signaling pathway.After then,siRNA-smad3 was introduced in order to block ActA/Smads signaling pathway. Protein and mRNA expression of caspase3 and the key sites of ActA/Smads signaling pathway were measured with the method of RT-PCR and western blot,and viability and apoptosis percentage were measured with the method of MTT,Hoechst33342 staining and AO/EB stain, in order to illustrate the effect of ActA/Smads signaling pathway and neurons transformed from PC12 cells subjected to OGD.Further, mechanism and action of ActA/Smads signaling pathway on ischemia and hypoxia neurons can be illustrated.Part 1:Culture and identification of neurons transformed from PC12 cells and establishment of OGD modelObjective:To culture and identify neurons transformed from PC12 cells, establish oxygen and glucose deprivation (OGD) model of activated PC12 cells.Methods:Cultured PC12 cells were activated by NGF and transformed into neurons.The neurons identified by morphology and immunofluorescence of anti NSE and MAP2. The culture were subjected to OGD injury using DMEM with no glucose and oxygen induced by NaS2O4, and were incubated in an anaerobic chamber which was refitted from a vacuum dryer. Viability and apoptosis percentage were measured with the method of MTT.Hoechst33342 staining after PC-12 cells were subjected to OGD injury.Results:PC12 cells were activated by NGF for 24h. show typical morphology, which account for 95% , immunofluorescence identification of NSE and MAP2 were strong positive. MTT shows viability of PC-12 cells subjected to OGD from 0h to 24h declined from 97.23% to 9.08%. and Hoechst33342 staining show apoptosis percengtage rose slowly before 12h,and at 12h rose sharply,reached the peak at 24h.Conclusions:Successfully culture and identify neurons transformed from PC12 cells, and set up oxygen and glucose deprivation (OGD) model of activated PC 12 cells.Part 2:Effect of ActA on ActA/Smads signaling pathway and apoptosis of PC-12cells subjected to OGDObjiective:Changes in basic components ActRâ…¡A,smad3,smad4 mRNA expression and apoptosis percentage of PC12 cells subjected OGD injury correlated with action of ActA was observed,in order to study the mechanism and effect of resistence to injury on ischemia and hypoxia.Method:In the basis of neurons transformed from PC12 cells and OGD model, in different concentration of exogenous ActA. Changes in viability and apoptosis were observed by MTT, Hoechst33324 staining respectively.the expression of mRNA of the key sites of ActA/Smads signaling pathway and caspase3 was measured by rt-PCR.Result:viability percentage was lower and apoptosis percentage was higher in OGD6h group than in control group. In different concentration of exogenous ActA, degree of injury to OGD decline more than in OGD6h(P<0.05), and there was a positive relativity between viability percentage and different concentration of exogenous ActA, and viability percentage in 100ng/ml exogenous ActA was highest among all groups. ActRâ…¡A,smad3,smad4 mRNA expression measured by rt-PCR were higher and Caspase3 mRNA expression were lower in ActA+OGD6h group than OGD6h and control group (P<0.05).Conclusion:exogenous ActA play a role in anti apoptosis and protecting neurons by ActA/Smads signaling pathwayPart 3:Establishment of Method of RNAi inhibiting the expression of smad3Objective:To observe the efficiency of plasmid vector transfection, and the gene silencing effect of RNAi on Smad3 in PC12 cultured in vitro.Methods:PC12 cells were used in the experiment. Short hairpin plasmid vector RNAi pGenesil-1A and B that could specially suppress Smad3 expression were constructed. The PC-12 were transfected using Lipofectamine 2000, and the expressions of EGFP were observed after 48h. The plasmid vector was chose which had higher transfection efficiency into PC12. contrast with control group and silencing control group. The expressions of smad3 mRNA were detected with rt-PCR at 3h,6h,12h,24h and 48h after reperfusion to observe the effect of smads-siRNA.Results:The passage culture of PC12 grew rapidly, and its appearance was the same as that of the primary cultures. The transfection efficiency of pGenesil-1B is higher than pGenesil-1A,48.62% and 11.24% respectively. After smad3 gene silencing, the expression of smad3 mRNA presented a significant reduction at any time, contrast with control group and silencing control group,and in the 24h group, the gene silencing effect was most obvious (p<0.05).Conclusions:smad3 RNAi pGenesil-1 could be transfected in passage cultured PC12 successfully using Lipofectamine2000. RNAi could inhibit the expression of smad3 in PC12, RNAi is a effective method to study Smad3.Part 4:Effect of SiRNA-Smad3 on ActA/Smads signaling pathway of PC-12cells subjected to OGDObjective:By observing change on ActRâ…¡A, smad3 and smad4 mRNA expression of the key sites of ActA/Smads signaling pathway in PC12 cells that were subjected to SiRNA-Smad3 and OGD, to probe into effect of smad3 on ActA/Smads signaling pathwayMethod:In the basis of Short hairpin plasmid vector siRNA-Smad3 constructed successfully and high efficiency of plasmid vector transfection into PC12 cells, after the signaling pathway was blocked by SiRNA-Smad3, the key sites mRNA andProtein expression of ActA/Smads signaling pathway were tested by rt-PCR and western blot.Result:ActRâ…¡A, smad3 and smad4 mRNA expression declined obviously after ActA/Smads signaling pathway was blocked with siRNA-smad3. smad3 and smad4 protein expression also declined obviously. ActRâ…¡A, smad3 and smad4 mRNA expression declined obviously in OGD group,and smad3 and smad4 protein expression also declined obviously contrast with control gruop(P<0.05).Conclusion:SiRNA-Smad3 can block ActA/Smads signaling pathway. Smad3 can play an key roles in ActA/Smads signaling pathway.Part 5:Effect of SiRNA-Smad3 on apoptosis of PC-12cells subjected to OGDObjective:By observing change on caspase3 in PC12 cells that were subjected to SiRNA-Smad3 and OGD, to probe into mechanism of effect of ActA/Smads signaling pathway on cells injury.Method:In the basis of Short hairpin plasmid vector siRNA-Smad3 constructed successfully and high efficiency of plasmid vector transfection into PC12 cells,after the signaling pathway was blocked by SiRNA-Smad3, viability percentage was measured by MTT and apoptosis percentage was detected by Hoechst33324 and AO/EB, and caspase3 was tested by realtime-PCR and western blot.Result:Caspase 3 mRNA and protein expression rised obviously after ActA/Smads signaling pathway was blocked with siRNA-smad3.Apoptosis percentage also rised obviously Caspase 3 mRNA and protein expression rised obviously in OGD group contrast with control gruop(P<0.05).Conclusion:SiRNA-Smad3 can block ActA/Smads signaling pathway. ActA plays a role in protecting neurons through ActA/Smads signaling pathway which mechanism perhaps is anti apoptosis and inhabinning caspasae3 protein expression. the up-regulation of ActA induced by activation of smad3 may play an foundmental role in the neuroprotective effect of ActA/Smads pathway.