Grant-funded Project Nr. 051/2001/C/2.LF Final Report
Project title:
In vitro model of the secondary cataract
Research leader:
RNDr. Richard Vytášek
Co-researcher:
MUDr. Gabriela Mahelková roz. Czakóová;
MUDr. Marie Vaňková
Period of project:
2001-2003
Overall grant:
504 000 CZK
Project Results
The first stage of project delt with isolation of porcine alpha-, beta- a gama-crystallins and after immunisation monoclonal antibodies against these proteins were prepared. Simultaneously the system of cultivation of porcine epithelial cells were developed (eg. the means of isolation of cells from lens a choosing of the best growth medium). The next period of project was targetted to characterisation of prepared monoclonal antibodies a development suitable SDS gradient polyacrylamide gel electrophoresis for separation of crystallins and their detection by immunoblotting. Crystallins from human health eye lens as well as from primary or secondary cataract were analysed by this system and it was observed that crystallins from secondary cataract are diametrically different from crystallins of primary cataract. Crystallins of health man are more similar to crystallins of primary cataract because main population of crystallins of health lens is also old. The next work delt only with materials extracted by various means (PBS and urea) from secondary cataract and from whole health lens a from proliferation area of eye lens. It was observed that the material from secondary cataract (with one exception) does not contain gama-crystallins. The amount of gama-crystallins in proliferation area of lens is lower than in other parts of health lens but it is detectable. The population of alpha-crystallins from secondary cataract exhibits two specific monomeric chains (19,0 and 21,4 kDa) that are not found in health lens. Finally beta-crystallins are highly covalently linked not only in health lens but also in relativelly young material from secondary cataract (of course in less extent) and this polymerisation of beta-crystallins is higher in less soluble urea extracted material. The study of cultivation of lens epithelial cells on various materials (glass, polystyrene, collagen I and IV) was performed and imunohistochemical analysis of differentiation (crystallins) and dedifferentiation (alpha-actin) markers was done. The growth of cells of primary culture is worse on glass. The attachment of cells of secondary culture is better on collagen I or IV but their next growth is the same on all tested materials. The primary cultures retained their differentiated state (no expression of alpha-actin) but the secondary cultures exhibited markers of dedifferentiation. The extend of dedifferentiation (production of alpha-actin) was lower when the cell density was higher and when cells were cultivated on collagen IV. The synthesis of crystallins by imunohistological detection was not observed (crystallins are mainly synthetised during differentiation of epithelial cells to fibrillar ones) but the synthesis of alpha- and beta-crystallins in primary culture cells during two months cultivation (morfologically the culture was similar to one type of secondary cataract – Elschnig´s pearls) were detected by imunoblotting. gama-Crystallins were not detected in this culture and this fact correlates with our findings during imunochemical analysis od secondary cataract. It seems that primary culture is suitable in vitro model of secondary cataract.