Laminin-5 is a component of preserved amniotic membrane.
OBJECTIVE
To determine if laminin-5 is retained in the matrix of cryopreserved human amniotic membrane tissue prepared for ocular surgeries.


METHODS
Amniotic membrane was solubilized in urea/SDS buffer. Constituent proteins were resolved by SDS-PAGE and laminin-5 content was determined by Western blot analysis using a panel of antibodies directed against the alpha3, beta3 or gamma2 chains of the molecule. Human corneal epithelial cells were seeded on amniotic membrane and cultured in the presence or absence of EGF. The cell-membrane construct was examined for laminin-5 content using Western blot analysis and immunofluorescence microscopy.


RESULTS
In preserved amniotic membrane the laminin-5 alpha3 chain is present in both the unprocessed (190-kDa) and processed (160-kDa) forms. The beta3 chain is found in the 145-kDa form. The gamma2 chain appears to be predominantly in the processed (105-kDa) form. Very little of the unprocessed form of the gamma2 chain (155-kDa) could be detected using immunoblot analysis. A similar distribution of laminin-5 was also present in extracts of corneal epithelial cells cultured on amniotic membrane. Immunofluorescence analysis of cells cultured on the membrane demonstrated polarization of laminin-5 at the cell-membrane interface.


CONCLUSIONS
The presence of both the unprocessed and processed forms of laminin-5 alpha3 and gamma2 chains in preserved human amniotic membrane suggests that when used as a substrate in ocular surgeries, this membrane may be capable of promoting corneal epithelial cell motility and adhesion. Regulation of the motile or adhesive function may lie with factors secreted by the corneal epithelium that populates the membrane following surgery.
Kurpakus-Wheater
M
M
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA. mkurpaku@med.wayne.edu
D000650:Amnion / Q000737:chemistry*; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002478:Cells, Cultured; D018920:Coculture Techniques; D003315:Cornea / Q000737:chemistry / Q000166:cytology; D015925:Cryopreservation; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007797:Laminin / Q000032:analysis*; D008856:Microscopy, Fluorescence; D014021:Tissue Preservation
D000911:Antibodies, Monoclonal; C400567:LAMC2 protein, human; D007797:Laminin; C095567:laminin alpha 3