Identification and clinical application of ectopic corneal epithelial cells in conjunctival epithelium

眼表面由2种生物学上不同的上皮细胞（角膜上皮和结膜上皮）覆盖;角蛋白12的表达目前被认为是角膜型分化的标志。我们研究了人球结膜上皮角蛋白12阳性细胞的生物学特性。应用免疫组化、原位杂交、免疫印迹、逆转录聚合酶链反应（RT-PCR）、荧光激活细胞分选（FACS）等方法检测人结膜角蛋白12（keratin 12）阳性细胞的分布。这些细胞的基因表达谱进行了引入扩增片段长度多态性（iAFLP）。为了确定干细胞或祖细胞的存在，进行免疫染色和集落形成测定。Western blotting、RT-PCR检测显示K12在结膜上皮中表达。免疫组化分析显示，K12阳性细胞主要聚集在结膜， ...更多信息 上皮细胞流式细胞仪分析显示，从下球结膜收集的结膜上皮细胞中有0.2-1.7%为角蛋白12阳性。iAFLP分析显示这些细胞的基因表达模式与角膜上皮细胞的基因表达模式高度相似。p63和ABCG 2在角蛋白12阳性细胞下方表达。一些集落形成细胞表达角蛋白12。这些结果有力地表明，角蛋白12阳性细胞似乎是异位居住，自我维持的角膜上皮细胞在结膜上皮。此外，我们研究了结膜上皮细胞是否可以用作角膜上皮细胞的替代来源。培养人结膜上皮细胞，制作复层上皮片。在全身施用免疫抑制剂的情况下，将该片材移植到兔角膜上。该片材在zenogeneic兔角膜上保持良好，并表现出角膜上皮细胞的几个特征，如角蛋白3/12表达。这些结果清楚地表明，人结膜上皮细胞可以在体外培养，并可用作替代角膜上皮细胞治疗严重眼表疾病的患者。少

The ocular surface is covered by 2 biologically distinct epithelia, the corneal- and the conjunctival epithelium ; the expression of keratin 12 is currently considered a hallmark of cornea-type differentiation. We investigated the biological features of keratin 12-positive cells in human bulbar conjunctival epithelium. Human conjunctival tissues were subjected to investigate the keratin 12 positive cells in conjunctiva by immunostaining, in situ hybridization, western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence-activated cell sorting (FACS). Gene expression profiling of these cells was carried out with introduced amplified fragment length polymorphism (iAFLP). To determine the presence of stem-or progenitor cells, immunostaining and colony-forming assay were performed. Western blotting, RT-PCR revealed that K12 was expressed in conjunctival epithelium. Immunostaining analysis showed that K12 positive cells reside mainly in clusters in conjunctiv … More al epithelium. FACS analysis showed that 0.2-1.7% of conjunctival epithelial cells collected from the inferior bulbar conjunctiva were keratin 12-positive. iAFLP analysis revealed that gene expression patterns of these cells were highly similar to that of corneal epithelial cells. p63 and ABCG2 were expressed beneath the keratin 12-positive cells. Some colony-forming cells expressed keratin 12. These results strongly indicated that the keratin 12-positive cells appear to be ectopically-residing, self-maintaining corneal epithelial cells in the conjunctival epithelium. Moreover, we investigated whether conjunctival epithelial cells can be used as alternative source for corneal epithelial cells. We cultivated human conjunctival epithelial cells and made stratified epithelial sheet. This sheet was transplanted onto rabbit cornea under systemic administration of immuno-suppressive agent. The sheet was well-maintained on the zenogeneic rabbit cornea and exhibited several features of corneal epithelial cells such as keratin3/12 expression. These results clearly indicate that human conjunctival epithelial cells can be cultured in vitro and may be used as alternative corneal epithelial cells for the treatment of patients with severe ocular surface diseases. Less