Abstract
Purpose: :
We aim to identify differentially expressed genes in Limbal epithelial crypt (LEC) which is a 120 microns solid cord of cells,arising from the undersurface of interpalisade rete ridges of limbal palisade of Vogt. This would be done in comparison to corneal, limbal and conjunctival epithelium with Gene ST 1.0 array to identify the markers,pathways related to these ocular surface (OS) regions.
Methods: :
RNA samples from lasermicrodissected (OS) epithelial regions were prepared from three pairs of human cadaver eyes. These were processed and hybridized to Gene ST 1.0 array chips. The chips were scanned to generate probe cell intensity data (CEL)files from the array images. The normalized data was then imported in Qlucore software. Differentially expressed gene list for each OS regions was created with ANOVA and two class comparison (filtration parameters of p value<0.05 and q values 0.2 to 0.6). Data mining was done. Validation of microarray data was performed with quantitative real time PCR.
Results: :
Of the 534 significantly expressed genes in LEC, 369 were down regulated and 161 up regulated.With highest number of down regulated genes LEC was the most dormant of all the OS epithelial regions.In Ingenuity pathway analysis LEC had enriched up regulated gene functions like cancer,hair,skin and hematological development.LEC was also enriched for metabolic pathways such as tyrosine metabolism, unlike cornea which was enriched for energy,protein, carbohydrate and fat metabolism.LEC was found to be significantly enriched for up regulated acute phase response signaling and stem cell pathways such as RAC ,PDGF and erythropoietin signaling pathways.Gene involved in embryonic stem cell pluripotency such as BMP3,AKT3,PDGFC were up regulated in LEC.LEC was enriched for GO terms related to quiescent stem cells such as regulation of DNA dependent transcription. Limbus was found to be enriched for epidermal keratinocyte and epithelial differentiation.GO term cell cycling was found to be enriched in cornea.Genes for self renewal such as PCNA, SULF2,GLS etc were found to be up regulated in cornea.
Conclusions: :
This study has shown that in normal OS health LEC is dormant and acts as reservoir of quiescent limbal stem cells. Enriched cell cycling with up regulated self renewal genes in cornea supports the evidence of transient cells (immediate stem cell progeny) in cornea. These could be responsible for maintaining epithelial turn over in normal healthy conditions of ocular surface.
Keywords: cornea: epithelium • gene microarray • cornea: basic science