Construction Of Recombinant Adenovirus Containing Human GFAP Promoter And P27Gene And Effects On The Gliosis

Objective: To construct adenovirus expression vector carrying the human GFAP promoter and p27gene and the control vector only carrying human p27gene.Methods:To construct and identify eukaryotic expression vector of pDC315-GFAP-p27-EGFP and pDC315-p27-EGFP by plasmid extraction, agarose gel electrophoresis, restriction enzyme digestion, connectivity, transformation and other genetic engineering techniques; To pack the recombinant adenovirus vector of Ad-GFAP-p27-EGFP and Ad-p27-EGFP expression vector by the AdMax adenovirus packaging systems and293cells; To identify recombinant adenovirus with enzyme digestion and PCR.Results:The eukaryotic expression vector of pGFAP-IRES2-EGFP-p27was identified using restriction enzyme digestion and sequencing; By restriction endonuclease and PCR analysis, the eukaryotic expression vector of pDC315-GFAP-p27-EGFP and pDC315-p27-EGFP was constructed successfully; After packaging, there was visible EGFP expression and cell pathogenic effect (CPE) in Ad-p27-EGFP group; CPE and weak expression of EGFP could be seen in Ad-GFAP-p27-EGFP group.Conclusion:Adenovirus expression vector carrying the human GFAP promoter and p27gene and the control vector only carrying human p27gene were constructed successfully by the adenovirus packaging system the AdMax, respectively. It may be helpful to further explore gene therapy approach for CNS diseases with excessive gliosis. Objective: To identify the effects of recombinant adenoviral vector of Ad-GFAP-p27-EGFP and Ad-p27-EGFP on proliferation of astrocyte in vitro and in vivo.Methods:For in vitro experiments, recombinant adenoviral vector of Ad-GFAP-p27-EGFP and Ad-p27-EGFP was infected into purified astrocytes. The expression of EGFP and p27, cell cycle and proliferation of astrocytes were examined by immunofluorescence, Western blot analysis, MTT assay and flow cytometry; Specificity of GFAP promoter was detected when recombinant adenoviral vector of Ad-GFAP-p27-EGFP was infected into neurons and endothelial cells.For in vivo experiments, recombinant adenovirus vector of Ad-GFAP-p27-EGFP and Ad-p27-EGFP was infected in rat brain following MCAO model. Expression of p27and VEGF, proliferation of astrocytes, neuron survival in perifocal tissue and behavior change were examined by immunofluorescence, Western blot, behavior NSS scores.Results:After recombinant adenoviral vector of Ad-GFAP-p27-EGFP and Ad-p27-EGFP was infected in astrocytes in vitro, the expression of EGFP was observed; The expression of p27was increased. The cell cycle and proliferation of astrocytes were inhibited; After recombinant adenoviral vector of Ad-GFAP-p27-EGFP was infected in neurons and endothelial cells, only weak expression of EGFP was visible; After recombinant adenovirus vector of Ad-GFAP-p27-EGFP and Ad-of p27-EGFP was infected into rat brain after MCAO model in vivo, the proliferation of astrocytes could be inhibited. Neuronal survival and neovascularization was enhanced in Ad-GFAP-p27-EGFP group. Finally, the NSS scores of rats were improved.Conclusion:The recombinant adenoviral vector of Ad-GFAP-p27-EGFP could inhibite gliosis effectively. Objective: To prepare improved three-dimensional heparan sulfate-collagen scaffolds and to assess the biocompatibility of heparan sulfate-collagen scaffolds and olfactory ensheathing cells.Methods:By improved dry and freezing procedures, three-dimensional sulfate heparin-collagen scaffolds was produced successfully. Three-dimensional structure of scaffolds was detected by scanning electron microscopy; Three-dimensional co-cultured system was established with improved three-dimensional sulfate heparin-collagen scaffolds and olfactory ensheathing cells in vitro. Cell proliferation was detected by MTT at2,6,10and14days; Olfactory ensheathing cells was labeled by CFSE at the appropriate concentration. The labeled cells in the scaffolds were tracked and observed directly under fluorescence microscopy. Adhesion and growth of olfactory ensheathing cells were also observed by scanning electron microscopy.Results:Improved three-dimensional heparan sulfate-collagen scaffolds showed three-dimensional microtubule structure, which could promote the adhesion, proliferation and process outgrowth of olfactory ensheathing cells. And the morphology of olfactory ensheathing cells showed bipolar and spindle. Processes of olfactory ensheathing cells grew out and extended at the same direction in three-dimensional culture system.Conclusion:The improved three-dimensional heparan sulfate-collagen scaffolds with olfactory ensheathing cells had good biocompatibility in vitro. Therefore, the improved three-dimensional heparan sulfate-collagen scaffolds may be used as a cell carrier. The scaffolds and olfactory ensheathing cells may form a three-dimensional compomers for nerve tissue engineering.