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Z. Barnard, G.R. Bushell, A.J. G. Apel, D.G. Harkin; Analysis of p63 Isoforms Expressed in Cultivated Autologous Limbal Epithelium for Clinical Treatment . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3029.
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To examine the RNA and protein expression of the various p63 isoforms in cultivated limbal epithelium produced for clinical use as autografts.
5 patients with limbal stem cell deficiency induced by varying causes had autologous limbal epithelium grown using the Green’s Method of keratinocyte expansion. Protein and RNA samples were extracted from cell pellets of the autologous epithelium and protein was examined via western blotting for p63 and keratin 3 expression using the 4A4 monoclonal and AE5 monoclonal antibodies respectively. Primers were designed to amplify all transactivating and all truncated p63 isoforms separately as well as both the transactivating and truncated p63 alpha isoforms specifically. Grafts of autologous epithelium were constructed for each patient by seeding the cultivated epithelium onto amniotic membrane stripped of its native epithelium and growing for a further 2 weeks. Duplicate grafts were also produced and sectioned for immunohistochemistry and stained for p63 and keratin 3.
All protein samples for the 5 patients expressed keratin 3. Each of the patient protein samples also produced 2 bands when stained with the 4A4 monoclonal antibody to p63, a major band at approximately 66KDa and another minor band at approximately 62KDa. RT–PCR demonstrated that all patient samples contained RNA for both transactivating and truncated classes of p63 isoforms as well as transcripts for both the transactivating and truncated p63 alpha isoforms specifically. All duplicate autologous grafts sectioned stained positively for keratin 3 and p63.
The cultivated limbal epithelium of patients suffering from limbal stem cell deficiency induced by varying causes exhibit very similar p63 isoform expression. Constructed autologous limbal epithelial grafts also exhibit similar expression of p63 and keratin 3 immunohistochemically. This work represents the first published account of p63 isoform expression in clinically used cultivated human limbal epithelial cells.
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