Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. 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 cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that 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. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.
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