ABSTRACT
Objective To evaluate the expression of human vascular endothelial cell growth factors 165 (hVEGF165) gene transfected into fibroblasts by recombinant adenovirus and study the repairing effect of this cells on radiated skin ulcer in rats.Methods The recombinant adenovirus with hVEGF165 was established and transfected to rat primary fibroblasts, and its expression of hVEGF165 in fibroblasts was identified with real-time PCR, immunocytochemistry and Western blot.Twenty four clean grade SD rats of were irradiated locally with 50 Gy γ rays to generate an animal model of radiation skin injury.The hVEGF165-transfected cells were injected to the irradiated site under rat skin 7 d post-irradiation.The therapeutic effects on the irradiated skin wound were evaluated through general observation as well as histological staining of HE.The expression of hVEGF in the irradiated skin tissue with fibroblasts injection was analyzed by Real-time PCR.Results The hVEGF165 gene was overexpressed in the transfected cells and approached to 88 373-fold bigger compared to controls transfected with blank vectors, and an extensive expression of VEGF in the cytoplasm of transfected cells was observed by immunohistochemistry.VEGF protein with the relative molecular mass of 23 000 was also detected in cell lysate by Western blot.The local skin ulcers in rats occurred about two weeks after irradiation.In the hVEGF165-transfected group, the average area of radiation-injured skin was 40.2 mm2, about 57％ less than that of the control group transfected with blank vectors so that the healing time was shorten by 6 days.The relative concentration of hVEGF mRNA in the skin tissue of rats injected with hVEGF165-transfected cells were 5.15-fold and 4.15-fold bigger compared to that of controls (t =3.385,3.220, P ＜ 0.05) at 3 and 7 d after administration.Conclusions The primary fibroblasts transfected with hVEGF165 gene could efficiently release VEGF to the irradiated skin tissue and promote the recovery of irradiation skin ulcers by shortening the healing time and thus enhanced the therapeutic effect on skin wounds.
ABSTRACT
Objective:To clone the fused gene of hVEGF_(165) and hirudin,construct a eukaryotic expression vector hVEGF_(165)-FH/pcDNA3.0 and express the vector in human endothelial cell strain. Methods: hVEGF_(165) cDNA fragment was amplified by PCR and fused hirudin peptide was synthesized by primer annealing. The fused hVEGF_(165 ) and hirudin gene was cloned into vector pcDNA3.0 to construct a new vector hVEGF_(165)-FH/pcDNA3.0 and the recombinant plasmid was confirmed by restriction enzyme and auto-sequencing. The correctly cloned plasmids were subjected to rapid in vitro transcription and translation identification. Liposome mediated, recombinant plasmids were used to transfect human endothelial cell strain and the products were identified. Results: hVEGF_(165)-FH was successfully fused and formed a 711 bp target fragment. The recombinant vector hVEGF_(165)-FH/pcDNA3.0 was successfully transformed into human endothelial cell strain which expressed the fusion protein. Conclusion: The recombinant vector hVEGF_(165)-FH/pcDNA3.0 can express fused protein in human endothelial cell strain, which paves a way for further study on the activity of the fusion protein and gene therapy.