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

眼表面覆盖着两种生物学上不同的上皮，角膜上皮和结膜上皮；目前，角蛋白12的表达被认为是角膜类型分化的标志。我们研究了人球结膜上皮中角蛋白12阳性细胞的生物学特性。采用免疫染色、原位杂交、免疫印迹、逆转录聚合酶链反应（RT-PCR）、荧光活化细胞分选（FACS）等方法对人结膜组织中角蛋白12阳性细胞进行研究。利用引入的扩增片段长度多态性（iAFLP）对这些细胞进行基因表达谱分析。为了确定干细胞或祖细胞的存在，进行了免疫染色和集落形成试验。Western blotting、RT-PCR检测显示K12在结膜上皮中表达。免疫染色分析显示K12阳性细胞主要聚集在结膜上皮内。FACS分析显示0.2-1.7%的下球结膜结膜上皮细胞角蛋白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高度相似。p63和ABCG2在角蛋白12阳性细胞下表达。一些集落形成细胞表达角蛋白12。这些结果强烈表明，角蛋白12阳性细胞似乎是位于结膜上皮内的异位、自我维持的角膜上皮细胞。此外，我们还研究了结膜上皮细胞是否可以作为角膜上皮细胞的替代来源。培养人结膜上皮细胞，制备成层状上皮片。在免疫抑制剂的作用下，将其移植到兔角膜上。该薄片在兔角膜上保存良好，并表现出角膜上皮细胞的一些特征，如keratin3/12的表达。这些结果清楚地表明，人结膜上皮细胞可以在体外培养，并可能作为替代角膜上皮细胞用于严重眼表疾病患者的治疗。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