In vitro methods, biomarkers and proteomics in eye research

Abstract

AbstractPurpose To understand biochemical pathways using cell culture models simulating certain aspects of eye disease combined with several modes of analysis. The use of primary cells and cell lines was explored as well as the use of laser capture micro dissection to reveal the roles of specific cell types of the ocular surface and meibomium glands.Methods Primary human conjunctiva cells were obtained from eye bank material as well as a conjunctiva cell line, IOBA cells. Additionally, limbal, conjunctival and eyelid tissues were obtained from human and mouse eyes. Full thickness cryostat tissue sections were made for laser microdissection (PALM Combi system) which were collected in 0.5ml tubes with 40ul or either Trizol for RTPCR or lysis buffer for protein extraction.Results Understanding the role of specific cells and their receptor specificities, the interaction of various epithelial cell layers with the environment in the conjunctiva and limbal regions has been difficult. The use of laser dissection has begun to clarify some of these relationships. In the meibomium gland neural receptors for NYP1, VIP1, SP and all five muscarinic receptors in the various cell types. In conjunctival tissue epithelial microregulators of found site specific distribution which may be related to the stem cell niche. Finally, laser dissection has allowed the properties of human basal layer limbal cells to be differentiated from the more anterior cells.Conclusion The ocular surface remains a challenge for understanding the interactions of the various epithelial cell types, the response to stress and the unique characteristics of the stem cells.