Graduation Year

2006

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

John R. Hassell, Ph.D.

Keywords

Stroma, Cornea, Insulin, Collagen/, Keratocan, Chromatography

Abstract

The corneal wound healing response involves the activation of keratocytes to proliferate from a quiescent phenotype. The mitogens that cause the initial transformation of the quiescent keratocytes to the active phenotype have not been identified. Even though serum is commonly used to replicate this in vitro, the cornea is avascular and therefore likely not exposed to serum. In the first part of this dissertation, a DMEM/F12 extract of corneal stromas was made and shown to stimulate keratocyte proliferation in both a dose-dependent and cell-density dependent manner. This extract contains mitogens that differ from the mitogens present in serum based on their effect on keratocytes and their biochemical characteristics. Culture in extract replicates in vitro the changes observed during the activation of keratocytes in the wound-healing phase.The corneal stroma contains an extensive extracellular matrix that consists primarily of collagens and proteolgycans.

This matrix is maintained and secreted by the keratocytes, cells with unique characteristics lost during the activation observed at wound healing. The second part of this dissertation aims to develop a defined culture medium that maintains the keratocyte phenotype during proliferation. Keratocytes were cultured in serum-free medium and the effect of the growth factors on the markers for the keratocyte phenotype determined. Only insulin was shown to stimulate cell proliferation in a consistent manner, while maintaining commonly accepted keratocyte markers. When this defined culture medium was supplemented with ascorbic acid to study collagen synthesis, a marked increase in both collagen synthesis and keratan sulfate proteoglycan accumulation was measured.

This newly developed culture medium, containing insulin and ascorbate, allows for cell growth, maintains the keratocyte markers, and could be used to study the native, non-activated keratocyte phenotype in culture.This dissertation shows that the culture media described herein replicate in vitro all the phenotypes observed during the corneal wound healing response in vivo. These culture media, in turn, could be used to obtain more knowledge about the different keratocyte phenotypes, and how they could be manipulated in culture. (329 words)

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