Hepatocyte Growth Factor (HGF) CDNA Cloning, Expression And Application In Vitro And In Vivo

Objective: The aim of this study is to establish nonviral gene therapy of Humanhepatocyte growth factor (hHGF) avoiding the viral vector-related side effects[1]. Bydetecting HGF expression in quality and quantity in human fibroblasts cutured in vitroand the application in vivo, our study provide an promising strategy for treatingalopecia areata and promoting hair regeneration in clinical therapy. Methods: Thewhole sequence of hHGF cDNA was cloned, conserted in the eukaryotic vector ofpTARGET plasmid, then the recombinant of pTARGET-HGF was constructed. WithLipofectamine2000, the pTARGET-HGF plasmid was transfected into human skinfibroblasts(HSF) cultured in vitro. In our laboratory we’ve previously constructed aplasmid pEGFP-N1-HGF which contains a report gene called green fluorescentprotein (GFP) and it was very intuitive to observe its transfection rate, so the besttransfection rate of pEGFP-N1-HGF was considerd for the pTARGET-HGF plasmidwhen optimizing the cell density, concentration of DNA and liposome. Western blotand enzyme-linked immunosorbent assay(ELISA) were applicated to determine HGFexpression both in quality and quantity. Subsequently pTARGET-HGF wassubcutaneously injected to alopecia areata murine. At last we investigated HGFexpression in vivo by HE staining and immunofluorescence staining. Result:1. Afterdigestion by BamHI and SalI and identified by agarose gel electrophoresis, two DNAsegments were obtained, that is,5.67kb and2.2kb in length, which is identical withthe pTARGET map.2. The fibroblasts cultured in vitro were transfected when80%ofthe cells were confluent with the recombinant pTARGET-HGF and the besttransfection was obtained with3×105cells/mL,0.8μg DNA and4.5μLlipofectamin2000, that is, the transfection were57.35±1.29%,52.47±1.26%and57.73±1.31%respectively.3. Western blot was applicated to detect HGF expression incell cultured in vitro and after collecting the supernatant, we found that the secretionof HGF in human skin fibroblasts48h after transfection was as much as5.76~10.74ng/9×105cells. Conclusion: We try to develop an promising strategy to treat alopeciaareata in gene level, constructed a vector containing HGF cDNA and applicated itinto animal model. We detected HGF expression both in vitro and in vivo and the growth of hair follicles were obviously improved after HGF injection. Therefore,HGF is an potent agent to promote hair regeneration.