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M.K. Gordon, P. Zhou, R.A. Hahn, C.A. Kistler, D.R. Gerecke, J.D. Laskin; Expression of Collagen IV Chain mRNAs by Immortalized Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1342.
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Purpose: It is imperative to establish the in vitro conditions that allow immortalized corneal epithelial cells to attain the synthetic capability of the in vivo corneal epithelium. With regard to extracellular matrix components, the uninjured in vivo corneal epithelium basement membrane contains type IV collagen made up of α3, α4, α5, and α6 chains, while α1, α2, α5, and α6 are found upon epithelial injury (Maguen, et al. Cornea 16:675-682, 1997). Immortalized corneal epithelial cells were plated on collagen I or on Matrigel, and examined for whether their expression of collagen IV alpha chain mRNAs resembled that of an injured or uninjured in vivo cornea. Methods: Primer pairs were designed to amplify the α1(IV), α4(IV), and α5(IV) collagen chain cDNAs. Immortalized corneal epithelial cells were grown on a collagen I substratum or on Matrigel. After reaching confluence, RNA was isolated from the cells and semi-quantitative relative RT-PCR was performed. Results were normalized to 18 S RNA content. Results: Cells plated on collagen I contain a large quantity of α1(IV) and α5(IV) collagen mRNAs, but undetectible amounts of α4(IV) mRNA, representative of what would be expected in an injured in vivo corneal epithelium. When plated on Matrigel, the immortalized corneal cells make α4(IV) and α5(IV) collagen mRNAs and small amounts of α1(IV) mRNA, mimicking the expression pattern of an uninjured donor cornea. Conclusions: These results strongly suggest that plating immortalized corneal epithelial cells on a Matrigel substratum allows them to express matrix components of an uninjured in vivo corneal epithelium, while cells grown on collagen I synthesize the type IV collagen chains typical of an injured cornea.
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