Cloning of Novel Epidermal Growth Factor (EGF) Plasmid for Gene Therapy on Diabetic Foot Ulcer.
10.4093/kdj.2008.32.2.131
- Author:
Hye Sook CHUNG
1
;
Chang Shin YOON
;
Min Jeong KWON
;
Mi Kyung KIM
;
Soon Hee LEE
;
Kyung Soo KO
;
Byung Doo RHEE
;
Jeong Hyun PARK
Author Information
1. Molecular Therapy Lab, Paik Memorial Institute for Clinical Research, Korea.
- Publication Type:Original Article
- Keywords:
Diabetes mellitus;
Epidermal growth factor;
Gene therapy;
Wound healing
- MeSH:
Animals;
Base Sequence;
Cell Culture Techniques;
Cell Line;
Clone Cells;
Cloning, Organism;
Diabetes Mellitus;
Diabetic Foot;
DNA;
Epidermal Growth Factor;
Furin;
Genetic Therapy;
Glycogen Synthase Kinase 3;
Humans;
Intercellular Signaling Peptides and Proteins;
Peptide Hydrolases;
Phosphorylation;
Plasmids;
Polymerase Chain Reaction;
Protein Isoforms;
Transfection;
Ulcer;
Wound Healing
- From:Korean Diabetes Journal
2008;32(2):131-140
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
BACKGROUND: Epidermal Growth Factor (EGF) is one of the important growth factors involved in the epithelialization during cutaneous wound healing. Peptide EGF has been used for the treatment of diabetic foot ulcer. But the inferiority of cost-effectiveness and the inconvenience of daily application might have restricted its wide clinical usage. EGF gene therapy could dramatically improve the efficacy and inconvenience through long-term expression and bypassing the EGF degradation by hostile non-specific proteinases expressed in the wound bed. METHODS: EGF DNAs were amplified via PCR. For the more effective secretion from the transfected cell, we inserted furin cleavage site into EGF plasmids. The efficacy of novel plasmid pbeta-EGF was verified by transfection into the various animal cell lines, and the biologic potency of expressed EGF was confirmed via phosphorylation of PI3K and GSK3beta by Western blotting. RESULTS: We tested various kinds of human EGFs. One of the human EGF isoforms, EGF(828) including a membrane-anchoring domain was successfully released as the mature EGF protein in the cell culture media. Also EGF plasmid including furin cleavage site showed more than 2-fold increased EGF expression compared with the sequence without furin cleavage site. CONCLUSION: In conclusion, these findings suggest that mature EGF could be released easily out of cells by modifying EGF DNA sequence. Our novel EGF plasmid DNA could markedly increase the efficiency of non-viral gene therapy for diabetic foot ulcer.
