摘要
Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells （MSCs）. Methods：The HGF cDNA was amplificated from the expression plasmid pCMV-HGF, and was subcloned into the adenovirus shuttle plasmid pDC316-IRESEGFP vector containing a green fluorescence protein （GFP） reporter gene. Virus Ad-HGF was produced by homologous recombination in HEK293 package cells. Bone marrow derived MSCs were harvested and cultured, and then were transduced with Ad-HGF. The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression. And HGF/MSCs were generated. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody. Results： The recombinant, named pDC316-HGF-IRES-eGFP, was digested with restriction enzyme, and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation. GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15 × 10^10pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 96.89% of HGF/MSCs were GFP positive. Peak concentration levels of hHGF（103ng/mL） in the cultured supernatants were detected on day 2 post-transduction, and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion：Our data demonstrated that the adenovirus expression＇vector system pDC316-HGF-IRES-EGFP has been constructed successfully, and their effective expressions also have been obtained in MSCs. This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.
Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells （MSCs）. Methods：The HGF cDNA was amplificated from the expression plasmid pCMV-HGF, and was subcloned into the adenovirus shuttle plasmid pDC316-IRESEGFP vector containing a green fluorescence protein （GFP） reporter gene. Virus Ad-HGF was produced by homologous recombination in HEK293 package cells. Bone marrow derived MSCs were harvested and cultured, and then were transduced with Ad-HGF. The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression. And HGF/MSCs were generated. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody. Results： The recombinant, named pDC316-HGF-IRES-eGFP, was digested with restriction enzyme, and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation. GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15 × 10^10pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 96.89% of HGF/MSCs were GFP positive. Peak concentration levels of hHGF（103ng/mL） in the cultured supernatants were detected on day 2 post-transduction, and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion：Our data demonstrated that the adenovirus expression＇vector system pDC316-HGF-IRES-EGFP has been constructed successfully, and their effective expressions also have been obtained in MSCs. This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.
基金
supported by National Natural Science Foundation of China(No.30671992)
a grant from the"135"Foundation of Jiangsu Province(No.135-10).
