The Research Of Transgenic CHO Recombined Mimetic Scaffolds Constructing Tissue-engineered Skin In Vitro

Objective:There are 150 million diabetic population all over the world at present, about 3%～6%of diabetic patients have a history of foot ulceration every year,to treat diabetic ulcers become a clinical problem that need to be solved urgently.In this research,the best property of PDGFs,PDGF-BB was selected as interest protein, constructed the enhanced green fluorescent protein eucaryotic expression vector carrying PDGF-B,transfected eukaryocyte and gained a cell strain which expressing interest protein steadily.Use natural macromolecule hydrogel material to compose 3D cell scaffolds loading transgenic cell constructed tissue-engineered skin, implementing PDGF-BB contacts the wound surface continuously and keeping a wet repair environment,to establish foundation for the next experiment of diabetic animal skin ulcers.Methods:Total RAN was extracted from HUVEC,two primers with restriction enzyme site of BamHI and NheI were designed and synthesized according gene sequence of human PDGF-B of Genbank and the multiple cloning site of pSELECT-GFPzeo-MCS Vector,human PDGF-B segment was successfully obtained by RT-PCR,then the PDGF-B gene was inserted into pMD 19-T vector to construct cloning plasmid pMD 19-T/rhPDGF-BB,after sequenced and identified,inserted the PDGF-B gene into pSELECT-GFPzeo-MCS vector to construct recombinant eukaryotic expression plasmid,transformed E.coli DH5αand obtained the recombinant pSELECT-GFPzeo/PDGF-BB plasmid,transfected it into CHO and used zeocin to gain a cell strain which expressing interest protein steadily,identified the interest protein by fluorescence microscope and immunocytochemistry,detected the protein quantity by ELISA,the effects of the interest protein on proliferation of NIH3T3 cell was essayed by MTT.Choosed natural macromolecule material chitosan and modified it by NAC to synthesize disulfide bond chitosan which sulfydryl contents is 321.4μmol/g,adopted 10 g/L concentrations to dissolve the compound into deionized water generating hydrogel,tested it's degree of swelling and cytotoxicity;used it loading transgenic CHO constructed tissue-engineered skin,scaned it's structure by SEM.Results:Agarose gel electrophoresis result of product of RT-PCR showed that specific proliferative band with relative molecular weight of 681 bp appeared in the prospective place,the double enzyme digestion of the recombinant cloning plasmid pMD 19-T/rhPDGF-BB corresponded with it,the result of sequence analysis was in agreement with the reported PDGF-B sequence in Genbank;The recombinant expression plasmid pSELECT-GFPzeo/rhPDGF-BB was confirmed correctly by enzyme digestion and sequence analysis;transformed E.coli DH5αand obtained the recombinant pSELECT-GFPzeo/PDGF-BB plasmid,transfected it into CHO and used zeocin to choose for 8 weeks,then gained a cell strain which expressing interest protein steadily,green fluorescence expressing after 8 weeks,immunocytochemistry displayed there were lots of positive proteins in the endochylema,the secreted interest protein quantity is 5.85μg/24 h/10~6 cells,and it obviously promoted NIH3T3 cell proliferation,it is statistical significance(p＜0.05).Choosed molecular weight is 300K Da,86%deacetylated chitosan and modified it by NAC to synthesize disulfide bond chitosan which sulfydryl contents is 321.4μmol/g,the compound is stable;dissolve the compound into deionized water generating hydrogel,degree of swelling of hydrogel is well,and has well biocompatibility(p＜0.05);used it loading transgenic CHO constructed tissue-engineered skin,SEM showed it have network structure,aperture well-distributed,cells stuck well.Conclusion:Succeeded constructed the enhanced green fluorescent protein eucaryotic expression vector carrying PDGF-B,transfected eukaryocyte and gained a cell strain which expressing interest protein steadily,the secreted interest protein obviously promoted NIH3T3 cell proliferation.Use natural macromolecule hydrogel material to compose 3D cell scaffolds loading transgenic cell constructed tissue-engineered skin,this scaffolds have well biocompatibility,aperture well-distributed,be good to cells growth,to establish foundation for the next experiment of diabetic animal skin ulcers.