Abstract
Purpose: :
Transplantation of human limbo-corneal epithelial (HLE) cells cultivated on amniotic membrane (AM) has been recognized as an effective stem cell therapy for the treatment of limbal stem cell deficiency. The purpose of this study is to use proteomic method in the hope of identifying factors that are involved in the progenitor cell-preserving mechanism.
Methods: :
Cell lysates from HLE cells cultured on intact AM (HLE/IAM) or on culture dish (HLE/dish) were subject to 2D gel electrophoresis, and proteins > 50% increase were analyzed by Ultraflex MALDI-TOF mass spectrometer. Annexin V staining and Western blot for cleaved poly ADP-ribose polymerase (C-PARP) were performed to study the anti-apoptotic effect of Hsp70-1 in Hsp70-1 SiRNA treated HLE cells after sublethal UVB irradiation. Finally, up- or down-regulation of deltaNp63α gene expression by plasmid transfection or addition of deltaNp63α SiRNA were performed to understand the causal relationship of deltaNp63α and Hsp70-1 expression.
Results: :
Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on intact AM, including Hsp70-1, Hsp-27, glutathione S-transferase, etc. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased C-PARP formation in hsp70-1-silenced, but not normal HLE cells. Over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA.
Conclusions: :
Hsp70-1 is over-expressed by HLE/IAM compared with HLE/dish, and deltaNp63α-directed Hsp70-1 over-expression may promote HLE progenitor cell survival on intact AM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.Financial disclosure: None
Keywords: proteomics • cornea: epithelium • apoptosis/cell death