Research TGF-Î'₃ Gene Recombination Adenovirus Compound The Marrow Mesenchyma Stem Cell On With Tissue Engineering Scaffold

PART ONE CLONING OF TARGET GENE TGF-β_3c2s2 AND GENERATING RECOMBINANT ADENOVIRUSObjective: Clone the target gene TGF-β₃ into the plasmid vector of adenovirus. Transfect the plasmid vector of adenovirus into the package cell HEK293 and generate the recombinant adenovirus expressing the target gene.Methods:Cut the plasmid containing TGF-β3, the separation after the enzyme, to purify and uses the directional clone into the pShuttle-EGFP, obtains pShuttle-EGFP-TGFβ₃, (adenovirus skeleton plasmid) changes over to the bacterium, in the bacterium with pAdxsi the homologous reorganization, obtains TGF-β3 reorganization adenovirus plasmid, inducts the HEK 293 cells by lipofectamine, produces the expression goal gene reorganization adenovirus carrier, determination reorganization adenovirus carrier titer by the method of dilutes.Results:â‘´TGFβ3gene clones enter in the adenovirus plasmid carrier to form the reorganization adenovirus plasmid pAdxsi-EGFP- TGFβ 3;⑵The reorganization adenovirus plasmid pAdxsi-EGFP- TGFβ3 inducts packing cell HEK 293 to form can express TGFβ3reorganization adenoviruses Ad- TGFβ3;⑶Time obtains compared to the dilution method determination has the high titer reorganization adenovirus Ad- TGFβ3.Conclusion: TGF-β3 gene clones to the reorganization adenovirus, success construction TGF-β3 gene recombination adenovirus carrier, the marrow mesenchyma stem cell compound chitosan scaffold's research for the further TGF-β3 base extension to build the good foundation.PART TWO RESEARCH TGF-Î’3 GENE RECOMBINATION ADENOVIRUS COMPOUND THE MARROW MESENCHYMA STEM CELL ON THE CHITOSAN SCAFFOLDObjective:â‘´Discussion the method of how to made the chitosan scaffold.⑵Disscussion the goal gene TGF-β3 revolutions to the marrow mesenchyma stem cell (MSCs) compound chitosan scaffold, the marrow mesenchyma stem cell's growth situation and the goal gene expression.Methods:â‘´Using the freezing drying method preparation chitosan scaffold,⑵Uses the density gradient pastes wall law isolated culture MSCs and observes the TGF-β3 transferred MSCs that cell's growth adherency situation by the fluorescence microscope and Western-Blot examination TGF-β3 gene extensions to dye MSCs TGF-β3 expression situations.⑶Oberserve the MSCs on chitosan scaffold situation by MTT law and the scanning electron microscope.Results:â‘´2% (w/w) the chitosan's solution use freeze-drying legal system results in the white sponginess chitosan scaffold.⑵The MSCs is pure and have nice figure, TGF-β3 genes can transfer effectively MSCs and Western-Blot obvious TGF-β3 transferred MSCs to have the masculine banding to appear.⑶Ad-TGF-β3 transfered the MSCs in the chitosan scaffold show great condition. Ad-TGF-β3 transfered the MSCs in the chitosan scaffold have no influence to cells in the scaffold. Ad-TGF-β3 transferred MSCs in the chitosan scaffold group have apperent activity than without scaffold group.Conclusion: MSCs on the chitosan scaffold show good growth adherency, the construction goal gene TGF-β3 transferred the marrow mesenchyma stem cell (MSCs) compound chitosan scaffold is the great organization project regeneration repair carrier.