Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting

[摘要] Objectives: The aim of this study is tocreate an ex vivo model to examine the expression of major heat-shockprotein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate70 (HCS70) at the mRNA and protein level in differentiating cornealcells from limbal stem cells (LSC) following air exposure. Methods: Limbal biopsies taken fromcadaveric normal human limbus were cultivated as explants on humanamniotic membrane (HAM) and plastic dish (PD). Corneal differentiationwas induced by air lifting for 16 days. The expression of putative LSCmarkers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers(keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90,and HSC70 were tested by RT–PCR, immunofluorescence, and flow cytometrypre- and post-air exposure. Fresh limbal and corneal tissues were usedas control groups. Results: Air lifting induced cornealdifferentiation with a decrease in the number of P63+ cellsand an increase in the number of K3+/CX43+ cells,which characterized transient amplifying cells (TACs). Moreover,denuded HAM provided a superior niche for LSC proliferation andphenotype maintenance in vitro. Additionally, we have evidence thatexpressions of HSC70 as well as HSP72 were enhanced through cornealdifferentiation and HSP90 post-air lifting in vitro and in vivo. HSP60,however, was not detected in either LSC or corneal cells, in vivo andin vitro. Conclusions: These results suggest thatcorneal differentiation following air exposure may regulate HSP72 andHSC70 expression. In addition, HSP72 and HSP90 may protect LSC andcorneal cells against oxidative stress.

[学科分类] 生物化学/生物物理