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

结膜上皮异位角膜上皮细胞的鉴定及临床应用

基本信息

批准号：
16390502
负责人：
KINOSHITA Shigeru
金额：
$ 8.77万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390502/
关键词：
keratin 12 corneal epithelium conjunctival epithelium transplantation gene expression tissue engineering cytokeratin 12 amniotic membrane immunostaining western blotting flowcytometry in situ hybridization

项目摘要

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

眼表被2个生物学上不同的上皮覆盖，角膜和结膜上皮覆盖。角蛋白12的表达目前被认为是角膜型分化的标志。我们研究了角蛋白12阳性细胞在人类鳞茎结膜上皮的生物学特征。通过免疫染色，原位杂交，蛋白质印迹，逆转录酶 - 聚合酶链反应（RT-PCR）和荧光激活的细胞分类（FACS），通过免疫染色，蛋白质印迹，蛋白质印迹，逆转录酶 - 聚合酶链反应（RT-PCR）（FACS），对人结膜组织进行结膜中的12个阳性细胞。这些细胞的基因表达分析是通过引入扩增的片段长度多态性（IAFLP）进行的。为了确定干细胞的存在，进行了免疫染色和菌落形成测定。 RT-PCR蛋白质印迹显示，K12在结膜上皮中表达。免疫染色分析表明，K12阳性细胞主要驻留在共同的簇中……更多的上皮细胞。 FACS分析表明，从下鳞茎结膜收集的结膜上皮细胞中有0.2-1.7％是角蛋白12阳性。 IAFLP分析表明，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阳性细胞下面表达。一些菌落形成的细胞表达角蛋白12。这些结果强烈表明，角蛋白12阳性细胞似乎是结膜上皮上皮细胞中生态保留的，自我维护的角膜上皮细胞。此外，我们研究了是否可以将上皮细胞用作角膜上皮细胞的替代来源。我们培养了人类结膜上皮细胞，并制成了分层的上皮片。在免疫抑制剂的全身给药下，将此片移植到兔角膜上。该薄片在靶流兔角膜上得到了很好的维护，并暴露了角膜上皮细胞（例如角蛋白3/12表达）的几个特征。这些结果清楚地表明，人结膜上皮细胞可以在体外培养，可以用作替代性角膜上皮细胞，以治疗严重的眼表面疾病患者。较少的

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A simple modification significantly reduces a background staining in immunostaining of corneal tissues using a biotinylated rabbit polyclonal antibody

使用生物素化兔多克隆抗体进行简单的修改即可显着降低角膜组织免疫染色的背景染色

DOI：
发表时间：
2005
期刊：
Experimental Eye Research 80
影响因子：
0
作者：
Kawasaki;S et al.;Kawasaki et al.;Hirai et al.;Kawasaki et al.
通讯作者：
Kawasaki et al.

Corneal cells for regeneration

角膜细胞再生

DOI：
发表时间：
2005
期刊：
Ernst Schering Res Found Workshop 54
影响因子：
0
作者：
Ichiro;Hashimoto;et. al.;Kawasaki et al.;Connon CJ et al.;Nakamura et al.;Kinoshita et al.
通讯作者：
Kinoshita et al.

Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium