The Role And Mechanism Of Cyclic-AMP Response Element Binding Protein In Mice With Oxygen Induced Retinal Neovascularization

Part one Construction,packaging and identification of recombinantadenovirus carrying small interfering RNA of mouse cyclic-AMP responseelement binding proteinPurpose:To construct a recombinant adenovirus carrying siRNA of CREB with enhanced green fluorescent protein tag.Methods:The sequence of CREB RNAi was designed with OptiRNAi Software,a online system of RNAi templet encoding,according to the design rules.These sense and antisense oligonucleotides were ligated to Age I and EcoR I and then annealed to form double strains of DNA that designated CREB(A/E).The double strains of siRNA targeting S1004 was insert into the linearized plasmid hU6-MCS-Ubi-EGFP that has been already digested with Age I and EcoR I to form the final shuttle plasmid hU6-CREB-Ubi-EGFP.After sequencing,the recombinant shuttle plasmid was cotransfected with the genomic plasmid pBHG lox ?E1,3Cre into HEK293 cells to obtain the recombinant adenovirus Ad-CREBRNAi-EGFP by AdMax system.The recombinant adenovirus was amplified by repeat infection of HEK293 cell and purified by Adeno-X Virus Purification Kit,and the the titer determination was done using the end-poin dilution method.Result:The recombinant adenovirus Ad-CREB-RNAi-EGFP was constructed successfully,which was confirmed by sequencing.The titer of recombinant adenovirus was 1.00 ×109PFU/ml.Conclusion:The recombinant adenovirus carrying small interfering RNA of cyclic-AMP response element binding protein with EGFP tag was successfully constructed.Part two The expression of cyclic-AMP response element binding protein inmouse model of oxygen-induced retinopathyPurpose:To establish the oxygen-induced retinopathy model in C57BL/6J mice by changing the oxygen concentration and explore the expression of CREB in mouse model with retinal neovascularization.Methods:170 7-day-old(P7)C57BL/6J mice were randomly divided into control group and OIR group.To establish the OIR model,mice from OIR group were exposed to(75±2)%oxygen for 5 days and then to the room air.In the control group,mouse pups were maintained in room air from P7-P17.Retinas from control and OIR group were collected for biochemical and morphological study.RNV was evaluated by quantification the area of vaso-obliteration and neovascularization in whole-mount immunofluorescent staining of mouse retina,as well as by counting the number of pre-retinal neovascular cells in pathological cross-sections.Mice from both the control and OIR group were sacrificed at postnatal day 12?14?17?19 and 21 respectively,then the retinas from different time point were collected to analyze the protein level and the mRNA level of CREB with Western blot and Real-Time PCR analysis.Results:In OIR group,the retinal neovascular tufts area and the vaso-obliteration area were both significantly larger than those in control group(P<0.05).The number of pre-retinal neovascular cell nuclei in retinas from OIR group were obviously higher than those in the control group(P<0.05).The protein and the mRNA level of CREB were significantly higher than those in the control group,and retinal CREB levels were positively correlated with the progression of retinal neovascularization.Conclusion:The CREB protein may be a pro-angiogenic factor in retinal neovascularization,by the reason of its level were positively correlated with the progression of retinal neovascularization in OIR mouse model.Part three cyclic-AMP response element binding protein gene silencing inhibitsthe retinal neovascularization in mouse model of oxygen-induced retinopathy Purpose:To investigate the effects on retinal neovascularization in OIR mouse model after silencing the CREB gene by intravitreal injection of recombinant Ad-CREB-RNAi-EGFP.Methods:138 7-day-old(P7)C57BL/6J mice were randomly divided into four groups including Normal group,OIR-CREB group,OIR group and OIR-GFP group.To establish the OIR model,mice from all groups except normal one were exposed to(75±2)%oxygen for 5 days and then to the room air.In the OIR-CREB group and OIR-GFP group,the OIR mice were given an intravitreal injection of 1?l of Ad-CREB-RNAi-EGFP or Ad-GFP at P12,and then returned to normoxia for the next five days.Retinas from all groups were collected for biochemical and morphological study.RNV was evaluated by quantification the area of vaso-obliteration and neovascular-ization in whole-mount immunofluorescent staining of mouse retina,as well as by counting the number of pre-retinal neovascular cells in pathological cross-sections.Mice from all groups were sacrificed at postnatal day 17,then the retinas were collected to analyze the protein level and the mRNA level of CREB with Western blot and Real-Time PCR analysis.Results:Fluorescence microscopy of retinal sections revealed that Ad-CREB-RNAi-EGFP was clearly present in the ganglion cell layer(GCL),inner plexiform layer(IPL),inner nuclear layer(INL),outer plexiform layer(OPL),and less strongly in the outer nuclear layer(ONL)of the retina.In the normal group without intravitreous injection,no positive amount of fluorescence was detected.In the OIR-CREB group,the retinal neovascular tufts area and the vaso-obliteration area were both significantly smaller than those in OIR group and OIR-GFP group(P<0.05).The number of pre-retinal neovascular cell nuclei in retinas from OIR-CREB group were obviously lower than those in the OIR group and OIR-GFP group(P<0.05).The protein and the mRNA level of CREB were significantly lower than those in the OIR group and OIR-GFP group(P<0.05).Conclusion:Ad-CREB-RNAi-EGFP gene transfer with intravitreal injection was efficient in OIR mouse model and the silencing of CREB ameliorates retinal neovascularization.Part four The mechanism of inhibitory effect on retinal neovascularization in mouse model of oxygen-induced retinopathy with cyclic-AMP response element binding protein gene silencingPurpose:To investigate the mechanism of inhibitory effect on RNV in OIR mice with CREB gene silencing,using the molecular biological techniques.Methods:104 7-day-old(P7)C57BL/6J mice were randomly divided into four groups including Normal group,OIR-CREB group,OIR group and OIR-GFP group.To establish the OIR model,mice from all groups except normal one were exposed to(75±2)%oxygen for 5 days and then to the room air.In the OIR-CREB group and OIR-GFP group,the OIR mice were given an intravitreal injection of 1?l of Ad-CREB-RNAi-EGFP or Ad-GFP at P12,and then returned to normoxia for the next five days.Mice from all groups were sacrificed at postnatal day 17,then the retinas were collected to analyze the protein level and the mRNA level of VEGF?HIF1??Bcl-2?Caspase-3with Real-Time PCR analysis and Western blot.Results:The protein levels of VEGF?HIFla?Bcl-2 in the OIR-CREB group were significantly lower than those in the OIR group and OIR-GFP group(P<0.05),while the protein level of Caspase-3 were significantly up-regulate in OIR-CREB group than those in the OIR group and OIR-GRP group(P<0.05).The mRNA levels of VEGF?HIF1??Bcl-2in the OIR-CREB group were significantly lower than those in the OIR group and OIR-GFP group(P<0.05),on the contrary,the mRNA level of Caspase-3 were significantly higher in OIR-CREB group than those in the OIR group and OIR-GRP group(P<0.05).Conclusion:CREB gene silencing ameliorates RNV in mouse model of OIR maybe through reducing the stimulation of VEGF/HIF-1? pathway,and which would lower the expression of Bcl-2 who were the downstream target of CREB.