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M.P. Fautsch, A.M. Vrabel, C.K. Bahler, D.H. Johnson; How Do Protein Expression Profiles from the Human Anterior Segment Culture Model Compare to the in vivo State? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3165.
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Purpose: For the human anterior segment culture model to be effective in studying the aqueous outflow pathway, it needs to represent the in vivo state, from both a histologic and molecular perspective. We determined if protein expression profiles from trabecular meshwork and effluent collected from anterior segments cultured with the usual medium (lacking growth factors and other constituents found in aqueous humor) differed from those cultured in medium containing serum. Methods: Normal human eyes obtained within 12 hours post-mortem were studied. Cultured anterior segments were prepared by removal of lens, iris, and vitreous; ciliary body was retained. Trabecular meshwork (TM) protein was obtained from meshworks dissected from fresh or cultured anterior segments (7 and 21 days). Effluents were collected daily from cultured anterior segments. Proteins were studied by 1-dimensional electrophoresis or by Westerns. The effect of changes in culture media was studied by culturing in media with and without 10% fetal bovine serum. Results: Proteins from perfusion cultured TM were similar overall to fresh TM at days 7 and 21, although several proteins appeared induced or repressed. Effluent media collected early in the culture period had high protein content that decreased dramatically within three days of culture. Myocilin expression diminished in both effluent and TM lysate within the first five days in culture. Interestingly, myocilin levels start to increase around day 9, but to levels that were less than 50% of Day 1 (n=5). The increase may represent adaptation to culture or stress. The addition of 10% fetal bovine serum maintained myocilin levels in effluent throughout the culture process. Conclusions: Protein profiles from fresh and cultured TM were fairly similar, although evidence for induction and repression of proteins was found. Significant changes in secreted proteins were found early in the culture process, representing a combination of proteins from the ciliary body, meshwork, and other anterior segment structures. The addition of supplements may be required for protein profiles to match those of the in vivo state.
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