Keratocyte Gene expression in the normal central cornea

Abstract

Purpose: : There is limited information on region specific gene expression in the human corneal stroma. In this study we used laser capture microdissection (LCM) to investigate the expression profile of extracellular matrix and adhesion molecules in the central human cornea.

Methods: : Frozen sections of human cornea without ocular diseases preserved in Optisol-GS solution were used to isolate the central corneal stroma by LCM. RNA was extracted from LCM captured stroma tissues. The RT2 Profiler PCR Arrays (SABiosciences) were used to examine the expression profile of extracellular matrix and adhesion molecules; Real-time quantitative (Q) PCR were used to quantify gene expression.The specificity of PCR products were determined by melting curve and agarose gel.

Results: : An average 10798 +/- 6896 keratocytes were isolated from the central corneal stroma by LCM. The RNA and cDNA were successfully prepared from the LCM-isolated keratocytes, and the quality was validated by the housekeeping gene β-actin.The gene expression profiling demonstrated that normal keratocytes in the central stroma reliably expressed 37 out of the 84 extracellular matrix and adhesion molecules represented in the array. The six mostly abundantly expressed genes and their relative expression against the housekeeping gene GAPDH were:COL6A2 (1524.88±712.60%); COL12A1(836.80±372.56%); TIMP2(643.75±275.82%); TGFB1(456.63+±196.77%); TIMP1(405.63±242.34%); ECM1(311.69±137.04%). Some of the more common ECM proteins in the stroma such as collagen I and other proteoglycans were not among the highly expressed genes.

Conclusions: : LCM followed by analysis of gene expression profiles is an excellent approach to discover the regional gene expression in the corneal stroma. These six highly abundant expressed genes could potentially function as a network of genes involved maintenance of corneal matrix integrity and clarity and could be potentially altered in corneal diseases.

Keywords: cornea: stroma and keratocytes • extracellular matrix • keratoconus