Molecular Analysis of Differentiation in Conjunctival Epithelium Cultivated on Human Amniotic Membrane by Real-Time RT-PCR

Abstract

Abstract: : Purpose:To analyze the effects of amniotic membrane transplantation (AMT) on mucin secretion and keratinization following ocular surface reconstruction. Methods: Conjunctival tissue samples were obtained from the fornix of normal eyes. Human conjunctival epithelium was cultivated on the epithelial side of the amniotic membrane (AM). SHEM medium was used until the cells became approximately 70% confluent, then exchanged to EpiLife^TM. Cells reached confluency under both air-lift and normal conditions. RNA was isolated from cultured conjunctival epithelium and Real-time RT-PCR amplification was performed in the presence of DNA double strand specific SYBR green dye for the detection of transglutaminase 1 and mucins mRNA. These were results compared with those cultivated on collagen-coated dishes. Results: Real-time RT-PCR showed that transglutaminase 1 mRNA expression was downregulated about 40% in culutivated on AM compared with collagen-coated dishes, and mucins mRNA expression was upregulated about 200% in culutivated on AM compared with collagen-coated dishes. Conclusions: AM may promote mucin secretion and differentitation of conjunctival epithelium, while suppressing keratinization.

Keywords: conjunctiva • transplantation • molecular biology