This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p = 0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p > 0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies.

中文翻译：

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角蛋白12 mRNA的表达可以作为角膜缘外植体培养的早期角膜标记。

本研究旨在通过角膜缘外植体培养物的转录分析来识别早期角膜标记和结膜上皮分化，并研究已知角膜和结膜分化标记的早期分化模式。从6名狮子国际角膜银行Saar-Lorloux / Trier-Westpfalz的供体获得2mm的角膜缘穿孔活检。将角膜外植体解剖成角膜和结膜活检切片。将活检样品的上皮侧向下放入12孔中。一旦生长的细胞达到汇合，就将它们收获。通过qPCR测量角膜分化标志物KRT12，KRT3，DSG1，PAX6，ADH7和ALDH1A1，mRNA的结膜标志物KRT19，KRT13和干细胞标志物ABCG2的mRNA表达。使用Western Blot评估KRT12和PAX6蛋白表达。结果表明，在活检角膜侧的结缔组织中，与结膜侧的结缔组织相比，KRT12 mRNA的表达明显更高（p = 0.0043）。与来自两个角膜缘活检的未生长细胞的标志物表达相比，其他分析标志物的mRNA表达没有显着差异（p> 0.13）。KRT12和PAX6 Western Blot分析显示，从两侧收获的细胞没有差异。总之，KRT12 mRNA可能是测量角膜缘和结膜祖细胞组成未知的角膜缘活检细胞角膜起源的标志物。KRT3，DSG1，PAX6，ADH7，ALDH1A1，KRT19，KRT13和ABCG2 mRNA以及KRT12和PAX6蛋白的表达不能帮助区分角膜与角膜缘活检的结膜细胞身份。