角膜结膜及泪器病             2074                                      PO0303

　　Gene Expression and Metabolism of Corneal Limbal Stem Cells on Different Growth Substrates

　　Shi-You Zhou MD1,2, Johann M. G. Reyes MD2, Roy S. Chuck MD PhD2, Jia-qi Chen MD1

　　Zhongshan Ophthalmic Center, 54 Xianlie Road, Guangzhou, China 510060

　　Objective: To investigate the metabolic properties of cultured corneal limbal epithelial cells and the associated gene expression changes on different substrates in order to disclose behavioral patterns of these cells when differentiating based on our previous study on mesenchymal stem cells which results were published on the journal of Stem Cells.
Methods: Limbal stem cells were cultured on collagen I and poly-d-lysine coated dishes and detected redox state by redox fluorometry and flow cytometry as well as the gene expression changes by real time quantitative reverse transcription polymerase chain reaction when cells became confluent.

　　Results: Autofluorescence ratios (reduced pyridines / oxidized flavoproteins) [redox fluorometry] of corneal limbal epithelial cells cultured on poly-d-lysine is 0.8638±0.2770, while 0.6895±0.6943 on collagen I substrates. Limbal stem cell intracellular oxidation state was detected by flow cytometry with fluorescent probe MitoTracker Orange CM-HM2 TMRos. The results of fluorescence strength showed that cells on collagen I coated substrate became more oxidized (108.06±13.91) than that on poly-d-lysine coated substrate (121.32±14.02). ABCG2 expression in limbal stem cells when cultured on poly-d-lysine is about 12.4 times than on collagen I substrates, ΔNp63α about 1.3 times as well as nestin about 1.8 times. But keratin 12 expressed much lower on poly-d-lysine, just 0.3 times, lower than half of gene expression on collagen I. There are significant different between these two groups either on autoflorescence ratio or flow cytometry as well as real time quantitative RT-PCR results (p<0.01).

　　Conclusion: Limbal stem cells may differentiate on collagen I rather than poly-d-lysine coated dishes. Poly-d-lysine is better for culturing corneal limbal stem cells to preserve genotype. When limbal stem cells differentiate, their redox state also changes correspondingly. Extracellular matrix may play an important role in promotion of corneal limbal stem cell differentiation.

　　Significence of this study: If redox situation between differentiating cells and stem cells is much different, limbal stem cells may be screened out by a simple way, autofluoresent microscopy.

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