Construction Of A Plasmid Vector Carrying Hepatocyte Growth Factor(HGF) Gene And Expression And Identification Of Its In Human Fibroblast

Objective: To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine t he effects of the hepatocyte growth factor (HGF) gene o n transfection and expression of human fibroblast cells in vitro. Methods: The full length c DNA o f hHGF, which was amplified from human mesenchymal stem cells mRNA by RT-PCR, was cloned into pEGFP-N1 vector to construct pEGFP-N1-HGF recombinant p lasmid. With Lipofectamine 2000 liposome, the recombinant plasmid pEGFP-N1-HGF was used to transfect t he c ulture human fibroblast c ells.After pEGFP-N1-HGF transfection, the following transfection conditions were optimized: cell density, DNA a mount, ratio of liposome and DNA. The t ransfection a nd expression of H GF i n hum an f ibroblast cells were tested by RT-PCR and Western Blotting, the level o f s ecreted HGF p rotein in s upernatant was d etermined w ith enzyme-linked immunosorbent assay (ELISA). Result: 1. Two DNA segments, about 4.7kb and 2.2kb in length, were obtained after digestion with BamHⅠand SacⅠ.The recombinant plasmid pEGFP-N1-HGF was identified by restriction endonuclease digestion and nucleotide sequencing. 2. The transfection efficiency was achieved with the following optimized conditions:At passage 2-5 later, 2×10~5 cells, 4ug DNA, 2.5:1 ratio for liposome and DNA, the results showed the highest transfection efficiency of human fibroblast cells ,which were 22.73±0.44%, 21.88±0.34%, 22.29±0.42%. 3. RT-PCR and Western-blotting proved that there was transcription of HGF gene in transfection c ells a nd t here w as e xpression of HGF in these c ells. When hHGF detected with ELISA in cell culture, there was a high level of hHGF expressed by transfected human fibroblast in 48 hours (5.9～7.55 ng/ 8×10~5 cells). Conclusion: The recombinant of pEGFP-N1-HGF is an effective expression vector. The recombinant plasmid pEGFP-N1-HGF can transcript and express in the transfected cells. HGF is thus a potent agent for strategies designed to promote therapeutic angiogenesis.