Purpose. Anterior chamber (AC) implantation has been studied as a useful experimental model, because anterior chamber is an immune-priveleged site, reportedly. We compared the expression of melanogenesis genes by reverse transcriptase-polymerase chain reaction (RT-PCR) method of human RPE cells cultured on laminin-coated collagen sheets and implanted into AC. Methods. Human RPE cells were cultured in minimum essential medium containing 10% fetal bovine serum with or without human basic-fibroblast growth factor (b-FGF). Human RPE cells were cultured on surface-modified polystyrene tissue culture plate. Cultures at passages 1 to 5 were seeded on laminin-coated collagen sheets. The origin of RPE cells was evaluated by cytokeratin immunocytochemistry. The sheets were implanted into AC of albino rabbit eyes, the other sheets from the same culture were continued to be fed every 3-4 days in vitro. After a month, collagenase digestion of the sheets (AC and in vitro) was made and the cells were counted. Specific primers against human tyrosinase, tyrosinase-related protein I (TRP-I) and tyrosinase-related protein II (TRP-II) were selected and RT-PCR was performed to the same amount of implanted RPE cells and in vitro cultured RPE cells. The specificity was confirmed by the digestion of specific restriction enzym. Results. Cytokeratin immunocytochemistry showed uniform staining of human RPE cells. Genes of tyrosinase, TRP-I and TRP-II were detected in human RPE cells cultured on collagen sheets and imp]anted to AC. The PCR products of melanogeneais genes in cultures made on collagen sheets and polystyrene plates showed selective difference. Conclusion,% I-This melhed is useful to determine in situ expression of specific genes of human RPE cells cultured on collagen sheet support which enables us the isolation of implanted cells. 2~Human RPE cells expressed melanogenesis genes a month after AC implantation Io albino rabbit, 3-The difference in melanogenesis genes expressiort between the RPE cells cultured on collagen sheets and implanted to AC. The PCR products of melanogenesis genes in cultures made on collagen sheets and polystyrene plates showed selective difference. Conclusions. 1-This method is useful to determine in situ expression of specific genes of human RPE cells cultured on collagen sheet support which enables us the isolation of implanted cells. 2-Human RPE cells expressed melanogenesis genes a month after AC implantation to albino rabbit. 3-The difference in melanogenesis genes expression between the RPE cells cultured on collagen sheets and on polystyrene plates could give further evidence of the importance of extracellular matrix proteins (collagen and laminin) and b-FGF for differentiation.
|Investigative Ophthalmology and Visual Science
|Published - 1996 Feb 15