The Effect Of Recombinant Adenovirus Containing GFAP Promoter And P27Gene On The Prolieration Of Astrocyte

Objective:Astrocytes play an important role in the formation of glial scar. This study was designed to construct Ad-pGFAP-p27-GFP recombinant adenovirus to specifically over-express p27protein in astrocytes, which will be used to inhibite astrocyte proliferation and glial scar formation.Methods:The recombinant adenovirus was constructed with the shuttle plasmid prepared before. The effect of recombinant adenovirus was assessed by GFP expression and CPE(Cytopathogenic effect). The overexpression of p27fusion protein was confirmed by immunofluorescence and western blot.Results:1) The recombinant adenovirus was constructed successfully.2) Recombinant adenovirus Ad-pGFAP-p27-GFP had specific effect on astrocytes with optimal MOI10.3) Immunofluorescence and western blot analysis showed that recombinant adenovirus Ad-pGFAP-p27-GFP could overexpress p27fusion protein in astrocytes.Conclusion:The recombinant adenovirus Ad-pGFAP-p27-GFP could specifically overexpress p27fusion protein in astrocytes. Objective:The above study has showed that recombinant adenovirus Ad-pGFAP-p27-GFP could specifically overexpress p27protein in astrocytes. Here we study the effects of recombinant adenovirus on the cell cycle and proliferation of astrocytes.Methods:EdU labeling and flow cytometry were applied to investigate the changes in cell cycle on astrocyte after Ad-pGFAP-p27-GFP treatment. The effect of Ad-pGFAP-p27-GFP on astrocyte proliferation was performed on a scratch injury model.Results:1) EdU labeling showed that recombinant adenovirus Ad-pGFAP-p27-GFP could delay the cell cycle of astrocyte and arrest them at Go-G1phase:At12h after infection, there was no obviously changes; At24h after infection, p27adenovirus could delay cell cycle compared with negative control(P<0.05); At48h after infection, p27adenovirus could delay cell cycle compared with that of negative control and null adenovirus (P<0.001);2) Flow cytometry showed that recombinant adenovirus Ad-pGFAP-p27-GFP could delay the cell cycle of astrocyte:At12h and24h after infection, there was no obviously changes; At48h after infection, p27adenovirus could delay cell cycle compared with negative control and null adenovirus (P<0.05);3) In starch injury model on cultured astrocytes, the p27adenovirus could suppress the proliferation of astrocyte near the scratch.Conclusion:Recombinant adenovirus Ad-pGFAP-p27-GFP could delay the cell cycle of astrocyte and block the proliferation.