INTRODUCTIONThe proteins found in tears play an important role in maintaining the ocular surface and changes in tear protein components may reflect changes in the health of the ocular surface. Proteomics provides a comprehensive approach for cataloguing all the proteins of the tear proteome, which will help to elucidate disease pathogenesis, make clinical diagnoses and evaluate the influence of medications on the structure, composition and secretion of tear proteins. In this study, an alternative proteomic strategy was investigated to explore the human tear proteome.

MATERIALS AND METHODSTear samples were obtained from patients who had pterygium and were collected on the first day and third day after pterygium surgery. Tears pooled from 6 patients were used in the analysis. Reverse-phase high-pressure liquid chromatograph (RPHPLC) was used as the first step to separate intact proteins into 21 peaks. Each fraction was then tryptic-digested and analysed by nanoLC-nano-ESI-MS/MS to characterise the protein components in each fraction.

RESULTSIn total, 60 tear proteins were identified with high confidence, including well-known abundant tear proteins, and tear-specific proteins such as lacritin and proline-rich proteins. Among them, proline-rich protein 5 was found for the first time in tear fluid. A large number of plasma proteins were also observed in tear fluid.

CONCLUSIONSThe results showed that the proteomic strategy used in this study was successfully applied to analyse tear proteome.

Eye Proteins ; analysis ; Humans ; Mass Spectrometry ; methods ; Proteome ; Tears ; chemistry

INTRODUCTIONIn this study, we have developed and optimised a novel gelatin-chitosan (GC) substrate for use as a cellular carrier for tissue-engineered conjunctival epithelium.

MATERIALS AND METHODSThe substrate was fabricated by casting and the mechanical properties of the substrate, including tensile strength and elongation, were measured. Using the MTT, cell proliferation assay with rabbit conjunctival fibroblasts, we optimised the G:C ratio to enhance cytocompatibility. Rabbit conjunctival epithelial cells were immunostained using monoclonal antibodies for keratin 4 and pancytokeratin to investigate the biological effects of the GC substrate on the proliferation and differentiation of epithelial cells.

RESULTSWe found that increasing the amount of gelatin resulted in an increase in elasticity (from 1:9 to 1:1 ratio), reaching a maximum (101.89% +/- 7.13%) at a ratio of 1:1. The MTT assay showed that the proliferation of conjunctival fibroblasts significantly increased from 0.068 +/- 0.017 to 0.177 +/- 0.011 (P = 0.014) as the gelatin was increased from 20% (1:4) to 50% (1:1). Additional studies using tissue-cultured conjunctiva explants showed that these explants grew well on the substrate, forming a multilayered epithelium. Cell morphology on this substrate was similar to that of cells grown on culture dishes alone. Positive staining of keratin 4 and pancytokeratin indicated that the substrate supported normal differentiation of conjunctival epithelial cells.

CONCLUSIONBy enhancing the proportion of gelatin, both the mechanical and biological properties of the chitosan substrate were improved. The results also suggest that this GC biomembrane may be a useful candidate for reconstructive tissue engineering of the conjunctiva.

Animals ; Biocompatible Materials ; Cell Proliferation ; Cells, Cultured ; Chitosan ; Conjunctiva ; cytology ; metabolism ; physiology ; Elasticity ; Epithelium ; physiology ; Gelatin ; Keratins ; metabolism ; Rabbits ; Tensile Strength ; Tissue Engineering