Journal of Ophthalmology / 2016 / Article / Fig 3

Research Article

Comparative Analysis of KnockOut Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

Figure 3

Gene expression and protein expression in human limbal epithelial cells. (a) Gene expression analysis of human limbal epithelial cells. PCR data shows that the cells cultured in both FAD medium and SR medium show high level expression of proliferation transcript PCNA in P1, P4, P7, and P10. The cells in both media show positive expression of cytokeratin 3 and cytokeratin 12, which implies their limbus origin. The positive expression of basal layer epithelial cell marker, cytokeratin 15, was also observed in both cultures. ABCG2 and ΔNp63α expression was detected in both cultures; their expression was observed to be decreased slightly after passage 7 (P7) in both culture media. The low level of connexin 43 (Cx43) expression was observed in both cultures and implied low level of epithelial cell differentiation in both media. (b) Quantitative real-time PCR analysis of human limbal epithelial cells cultured in SR and FAD media. Real-time PCR was performed on epithelial cells cultured using both FAD and SR media. Housekeeping gene GAPDH was used as an internal control. Analysis of the limbal epithelial stem cell markers p63 and ABCG2 mRNA found that there was no significant difference (, ) in expression between epithelial cells grown in FAD and SR media. Real-time analysis of the corneal epithelial differentiation marker cytokeratin 3 and connexin 43 was performed. All these markers were barely detectable in both FAD and SR cell cultures, and, surprisingly, there is no significant difference (, ) of expression level between these two cultures. All the real-time PCR experiments were carried out in triplicate. (c) Western blot assay of human limbal epithelial cells cultured in FAD and SR media. The expression of proliferation marker (PCNA) and potential epithelial stem cells markers (p63 and ABCG2) was evaluated using western blot. Using monoclonal antibody clone 4A4, the p63 protein (70 KD) was detected at high expression level in SR medium. The expression of ABCG2 was detected in each passage of cells cultured in SR medium. PCNA, the cell nucleus antigen, a cell proliferation marker, was also detected in epithelial cells cultured in SR medium. (d) The expression of ABCG2, p63, and PCNA was quantified and plotted using semiquantitative intensity measurement. The blot density of ABCG2, p63, and PCNA was measured and normalized to the level of GAPDH.

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