Screening Possible Scar-Collagen Inhibitors on TGF-β1 Treated Dermal Fibroblasts
Graduation Date
5-2018
Document Type
Honors Thesis
Degree Name
Bachelor of Science
Department
Natural Sciences and Mathematics
Director of the Honors Program
Gigi Gokcek, PhD
First Reader
Diara Spain, PhD
Second Reader
Tyler Johnson, PhD
Abstract
When cells are wounded, functionality is decreased due to collagen being produced at high levels. The accumulation of collagen is called scarring or fibrosis. To simulate collagen levels during scarring primary dermal fibroblasts were treated with transforming growth factor beta I (TGF-β1) before the addition of test compounds. TGF-β1, a cytokine, is a known inducer of scar collagen production by fibroblasts. Anti-fibrotic effects of test compounds were screened and evaluated in vitro by measuring amounts of collagen produced at the protein level. To measure secreted collagen as part of the extracellular matrix monoclonal antibodies against collagen I and III were used. Collagen I is the most abundant type in the human body. III is present along with type I in connective tissue, blood vessels, and skin. Anti-fibrotic effects were measured in a fluorescent microplate assay for secreted collagen I and III and confirmed by confocal microscopy for collagen I. MTT assays confirmed anti-collagen effects from the test compounds were not due to decreased cell viability. From the results of these tests epigallocatechin gallate (EGCG) and nintedanib show promising decreases in collagen production in a concentration dependent manner in comparison to the control, illustrating the usefulness of the assay for screening scar antagonists.
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