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

眼表被两种不同的生物上皮、角膜上皮和结膜上皮所覆盖；角蛋白12的表达目前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。采用免疫组织化学染色、原位杂交、Western blotting、逆转录聚合酶链式反应(RT-PCR)和荧光激活细胞分选(FACS)等方法对人眼结膜组织中角蛋白12阳性细胞进行检测。采用引入的扩增片段长度多态性(IAFLP)技术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结果显示K12在结膜上皮细胞中有表达。免疫组织化学染色分析显示，K12阳性细胞主要分布在结膜…中更多的铝上皮细胞。FACS分析显示，从下球结膜采集的结膜上皮细胞中有0.2-1.7%为角蛋白12阳性。IAFLP分析表明，这些细胞的基因表达谱与角膜上皮细胞高度相似。角蛋白12阳性细胞下方有p63和ABCG2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驻留的、自我维持的结膜上皮细胞。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，制成层状上皮片。在全身应用免疫抑制药的情况下，将该膜移植到兔角膜上。该膜在青春兔角膜上保持良好，并显示出角膜上皮细胞的几个特征，如角蛋白3/12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，可以作为替代角膜上皮细胞用于治疗严重的眼表疾病患者。较少

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