Purification of recombinant fusion polypeptide hEGF-AWRK6 and effect on wound healing and infection of burn model mice / 生物工程学报

To test the therapeutic effect of recombinant fusion polypeptide hEGF-AWRK6 (EK) on burn infection of model mice. EK6 was expressed and purified with Escherichia coli expression system, and the Ⅱ degree burns and Pseudomonas aeruginosa infection model mouse were established. Experiment group was treated with EK (30 mg/L), and the control group was treated with PBS, gentamicin (30 mg/L), burn ointment (10 mg/L). The wound healing rate and colony count were calculated. Wound and surrounding skin were taken for HE staining and collagen western-blot analysis, and the wound pathological changes were observed after 10 days of drug delivery. The results showed that fusion peptide EK was successfully expressed and purified with significant antibacterial activities against Pseudomonas aeruginosa. Compared to the control group, the colony count (CFU) of the wound surface in EK mouse had a remarkable decrease (P<0.01) and healing rate had a significant increase in group EK6 (P<0.01). Pathological analysis result showed that compared to the control group, wound dermal cells in group EK arranged regularly, had more hair growth and a faster epithelization. These results indicated that the fusion peptide EK would be a good candidate for the drug development for the treatment of burning wounds.

Optimal Concentration of hEGF for Epithelial Healing in Experimental Corneal Alkali Wounds

PURPOSE: An optimal concentration of hEGF to enhance epithelial healing was determined in the rabbit eyes after alkali wounds. METHODS: Alkali-injured corneas (=5.5 mm, 1N NaOH, 60s) were treated with 0 (control), 2, 5, 10, and 50microgram/ml hEGF 3 times per day. The measurement of epithelial healing rate, transmission electron microscopy and immunohistochemical observation were performed after 7 days treatment. RESULTS: No significant difference was observed in the rates of epithelial healing between control and each hEGF-treated group. Among the tested concentrations, 5microgram/ml hEGF induced the most active proliferation of basal cells and 50microgram/ml hEGF remarkably produced a vascular ingrowth to the central wound area. The number of epithelial layers was increased by hEGF in a concentration-dependent manner. CONCLUSIONS: A low concentration of hEGF may selectively enhance epithelial healing without inducing corneal neovascularization. The optimal concentration of hEGF for the stimulation of basal cell proliferation appears to be 5 microgram/ml in rabbit corneal alkali wounds.

Effect of Topically Applied Human Epidermal Growth Factor on Stromal Keratocyte Activity in Experimental Corneal Alkali Wounds

PURPOSE: The Influence of topically applied human epidermal growth factor(hEGF) on stromal keratocyte was evaluated in the experimental corneal alkali wounds. METHODS: Corneal alkali wounds were induced by applying a 5.5 mm round filter paper soaked in 1N NaOH onto the center of rabbit cornea for 60 seconds. The eyes were treated topically with 5 micrometer/ml hEGF(DaeWoong Pharma. Co. Ltd. Seoul, Korea) or PBS, 3 times per day. After 3- and 28-days treatment, the corneas were processed for transmission electron microscopy(TEM). RESULTS: After 3-days treatment, the basement membrane integrity was maintained and the stromal keratocyte remained damaged in both groups. However, after 28-days treatment, the basement membrane was disrupted and the keratocytes were activated in both groups. CONCLUSIONS: The findings of present study indicated that the optimal concentration of hEGF which is known to stimulate the epithelial healing was failed to affect the keratocyte activities as long as the basement membrane integrity was maintained.

Optimal Concentrations of hEGF for Epithelial and Endothelial Healing in Corneal Alkali Wounds: An Organ Cultured Study

Optimal concentrations of hEGF for epithelial and endothelial healing after alkali-injury were determined in an organ cultured system. By using a 5.5 mmround filter paper, corneal alkali wounds (1N NaOH, 60sec.) were induced in the rabbit eyes and then the whole cornea with 1 mmscleral rim were excised.The excised corneas were immersed in the Eagle`s minimum essential medium supplemented with 5% Dextran, 1%antibiotics and different concentrations of hEGF (DaeWoong Pharma.Co.Ltd. Seoul, Korea), i.e., O (control), 0.01, 0.1, 1.0, 10, 100ng/ml. After tryphan blue staining, morphometric analysis was performed at 1, 2, 3, 4 and 6 days time periods. The healing rates were calculated by using an image analysis system. After 7 days, the corneas were processed for an immunohistochemical observation of proliferating cell nuclear antigen (PCNA) positive cells in the re-surfaced epithelium. Epithelial re-surfacing was significantly enhanced by the hEGF treatment at a concentration of 1.0ng/ml. The thickness of regenerated epithelium appeared to be increased in a dose-dependent man-ner. The rate of endothelial healing was increased at the concentration of 100ng/ml. The results indicate that epithelial healing was stimulated at about 100 times lower concentration of hEGF than endothelial healing.