角膜上皮细胞Pax-6基因表达分析(英文)

Expression of Pax-6 gene in corneal epithelial cells in Vitro

目的 检测体外培养角膜上皮细胞Ｐａｘ－６基因的达，探讨维持角膜缘干细胞特性的基因因素。 方法 地高辛标记ｃＤＮＡ探针进行ＤＮＡ印迹杂交和蛋白质免疫印迹，检测培养的角膜上皮细胞中Ｐａｘ－６基因表达状况。 结果 ＥｃｏＲ Ⅰ酶切角膜缘上皮细胞中９．１ｋｂｐ的ＤＮＡ片断与探针序列相同，其中原位和原代培养细胞、传第１代明显测出，角膜中央上皮细胞的原位和原代细胞中也能测出。Ｗｅｓｔｅｒｎ Ｂｌｏｔ发现抗血清１１认识相对分子质量为８１×１０３和５５×１０３两种角膜上皮细胞蛋白，抗血清１３认识６０×１０３的角膜上皮细胞蛋白，原位和原代、传第１代角膜缘干细胞和角膜中央上皮细胞均能够测出，角膜缘传第３代细胞和角膜基质细胞不表达。 结论 角膜缘干细胞和角膜中央上皮细胞均具有Ｐａｘ－６基因，该基因的正常表达是维持角膜上皮细胞特性的必要条件。

Objective To study gene expression of Pax-6 in cultured corneal epithelial cells. Methods The expression of Pax-6 gene in corneal epithelial cells in vitro was demonstrated by Southern and Western blots. Results A 9.1kbp genomic DNA from corneal epithelial cells was positively hybridized with the probe of Pax-6 in Southern blot.Pax-6 was positive in first and second passage of limbal epithelial cells and in primary culture of central corneal epithelial cells in situ .The antisera ll which was against the paired domain of Pax-6 could recognize all of the 81kDa and 55kDa proteins in corneal epithelial cells, but the antisera 13 which was against the homeodomain of Pax-6 could directed against a 60kDa protein in primary and first passage cul- ture of the limbal and central epithelial cells ,but it was nagetive in the third passage culture of limbal epithelial cells and stromal cells. Conclusions The limbal and central corneal epithelial cells are positive for the gene of Pax-6.The normal expression of Pax-6 gene is vital for maintaining the corneal epithelial cells characteristics.