THE EFFECTS OF DOUBLE STRANDED OLIGODEOXYNUCLEOTIDE ON COLLAGEN PRODUCTION IN A WOUND HEALING MODEL
Open Access
- Author:
- Rosensteel, Shawn Michael
- Graduate Program:
- Laboratory Animal Medicine
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Ronald Paul Wilson, Thesis Advisor/Co-Advisor
H Paul Ehrlich, Thesis Advisor/Co-Advisor
Xuwen Peng, Thesis Advisor/Co-Advisor
Mark Kester, Ph D, Thesis Advisor/Co-Advisor - Keywords:
- oligodeoxynucleotide
collagen
wound healing
wound - Abstract:
- Type I collagen is an integral part of dermis, granulation tissue and scar. Collagen deposition is promoted by TGF-β1 a member of a family of pro-fibrotic cytokines, which promotes fibroblast chemotaxis, proliferation, transformation into myofibroblasts, and the synthesis of collagen. We hypothesize that inhibiting the transcription of the COL1A1 gene, type I collagen will be suppressed early in the wound healing process. In this study, local injection of a double stranded oligodeoxynucleotide (dsODN) decoy containing the TGF-β regulatory element found in the distal promoter of the COL1A1 gene, is evaluated in a rat model of wound healing. The effect of reducing the synthesis of type I collagen on wound healing is evaluated by the analysis of type I collagen, type III collagen, and extra domain A (ED-A) fibronectin synthesis. Analysis of fibroblast proliferation, and angiogenesis as evaluated by vascular endothelium was performed. Fluorescent microscopy was used to assess granulation tissue responses to dsODN uptake by the target cells fibroblasts and myofibroblasts. Dot Blot analysis of EDA-fibronectin levels to type I and type III collagen levels were compared in implants treated with 10 nM dsODN, type I (p=0.02)and type III (p=0.01). Collagen levels were significantly decreased compared to controls. Treatments with 3 nM and 1 nM dsODN did not produce significant changes in the levels of type I, type III collagen or fibronectin. Compared to scramble dsODN decoy therapy, histology of decoy treated implants revealed increased cellular density due to decreased connective tissue deposition. When treated implants were stained with Sirius red and viewed under polarized light, little collagen appeared between fibroblasts in treated implants. These collagen fiber bundles demonstrated a fine green birefringence, consistent with immature collagen fibers. Fluorescently labeled dsODN was found in the nuclei of both fibroblasts and myofibroblasts. In conclusion, dsODN therapy was successful at decreasing type I collagen and also had effects on type III collagen in a wound healing model. Histology revealed increased cell counts corresponding to areas of decreased collagen content.