Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were use...Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing展开更多
A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid D...A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was de-tected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereas no therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were ob-served to different extent on healed wound treated with Ad-HGF. Histopathologic examination re-vealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amo展开更多
基金theStateHigh TechnologyDepartmentProgram (No 2 0 0 1AA2 170 61)partlybyagrantfromtheBiologicalEngineeringDepartment+1 种基金FarEastAreaofAmericanBaxterHealthcareProductsCo Ltd USA
文摘Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing
基金supported by the State High Technology Development Program(Grant No.2001AA217061).
文摘A complementary DNA (cDNA) encoding human hepatocyte growth factor was intro-duced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homolo-gous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities to human hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was de-tected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF, the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that of the normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereas no therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were ob-served to different extent on healed wound treated with Ad-HGF. Histopathologic examination re-vealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed. In Sirius red-stained sections the amo
