Abstract
P 073
The mRNA expression of the complement factor H is upregulated in proliferating cultured human retinal pigment epithelial cells
Norbert Kociok1, Antonia Joussen2
1Labor für Exp. Ophthalmologie, Universitäts-Augenklinik Düsseldorf, 2Universitäts-Augenklinik Düsseldorf
Purpose
We used early passages of cultured human retinal pigment epithelial cells (RPE) as a model system for identifying differentially expressed genes without further prerequisites. The gene and protein expression of the identified gene in RPE cells was analyzed subsequently.
Methods
We used a differential expression analysis (DEmRNA-PCR) to find differentially expressed genes in early passages of cultured human RPE cells. The detected mRNA was identified by sequencing and its differential expression in RPE cells was verified by semi-quantitative RT-PCR. The expression of the identified protein in vitro and in surgically removed epiretinal membranes was demonstrated by western blotting and immunocytochemical analysis.
Results
With DEmRNA-PCR a decreased expression of a band at approximately 530 bp was detected in human RPE cells of passage 3 (P3) compared to P0. This band was identified as part of the human complement regulatory factor H, a cofactor to complement factor I. The mRNA expression of both regulatory proteins of the complement system was confirmed in freshly prepared human RPE cells and in cultured cells from passage 0 to passage 8 by gene specific reverse transcription-PCR. The expression of both proteins in cultured RPE cells was verified by western blotting. The demonstration of both proteins in surgically removed epiretinal membranes was verified by immunohistochemistry.
Conclusions
With the detection of the differential expression of the regulatory factors H and I of the complement system in cultured RPE cells by a technique without any prerequisites the importance of these factors in RPE cells was demonstrated. Our results may indicate an equal importance of the complement system in proliferative retinopathy in addition to its known role in age-related macula degeneration.
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