Elucidation of gene regulation mechanism by which corneal epithelial cells achieve their specific differeatiation status, especially those regarding to corneal epithelial cell-specific transcription factor

阐明角膜上皮细胞实现其特定分化状态的基因调控机制，特别是与角膜上皮细胞特异性转录因子有关的基因调控机制

基本信息

批准号：
18390472
负责人：
KINOSHITA Shigeru
金额：
$ 11.28万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Keratin 12 is a gene that is specifically expressed in corneal epithelial cells. In this study, we investigated whether such a tissue-specific expression is controlled by the genomic methylation status. First, we evaluated keratin 12 gene expression among various types of human keratinocyte cells. As has been reported by many researchers, keratin 12 was only expressed in in vivo corneal epithelial cells. Surprisingly and notably, subcultured primary human corneal epithelial cells (hCEC) and immortalized hCEC did not express keratin 12. We then treated the immortalized hCEC by 5-aza-cytidine, a demethylation agent and TSA, a histone deacetylase inhibitor to see whether such a tissue-specific gene expression of keratin 12 involved an epigenetic gene regulation mechanism. We subsequently discovered that those cells express keratin 12. Next, we performed bisulfite sequencing analysis to see the methylation status of keratin 12 among various kinds of in vivo keratinocyte cells. As a result, we found that keratin 12 was in the hypomethylated status in in vivo corneal epithelial cells, while other keratinocyte cells exhibited hypermethylation of keratin 12. Also, the subcultured primary hCEC (P6, P7, and P8) and the immortalized hCEC exhibited hypermethylation of keratin 12. We then tested the hypothesis that the culture process may affect the genomic methylation status. We performed repeated culture of hCEC from in vivo to P6 and subjected each of these cells to bisulfite sequencing analysis. We found that the keratin-12 methylation rate was gradually increased by the number of passage. Finally, we evaluated the methylation status of the limbal basal epithelial cells, which have been reported to contain the stem cells of the corneal epithelial cells: We found that keratin 12 was in the hypermethylated status in these cells, suggesting that keratin 12 may be demethylated when the limbal basal cells are differentiated into corneal epithelial cells.

角蛋白12是一种在角膜上皮细胞中特别表达的基因。在这项研究中，我们研究了这种组织特异性表达是否受基因组甲基化状态控制。首先，我们评估了各种人角质形成细胞中角蛋白12基因的表达。正如许多研究人员报道的那样，角蛋白12仅在体内角膜上皮细胞中表达。 Surprisingly and notably, subcultured primary human corneal epithelial cells (hCEC) and immortalized hCEC did not express keratin 12. We then treated the immortalized hCEC by 5-aza-cytidine, a demethylation agent and TSA, a histone deacetylase inhibitor to see whether such a tissue-specific gene expression of keratin 12 involved an epigenetic gene regulation 机制。随后，我们发现这些细胞表达角蛋白12。接下来，我们进行了硫酸盐测序分析，以查看各种体内角质形成细胞中角蛋白12的甲基化状态。结果，我们发现角蛋白12处于体内角膜上皮细胞中的低甲基化状态，而其他角质形成细胞则表现出角蛋白12的高甲基化。此外，亚培养的原发性HCEC（P6，P7和P8）以及不成熟的HCEC均具有超含量的促成促成的促成促成的凯辛碱基化的过程。基因组甲基化状态。我们从体内到P6进行了HCEC的重复培养，并将这些细胞中的每一个都进行了亚硫酸盐测序分析。我们发现角蛋白12甲基化速率随通道数量逐渐提高。最后，我们评估了缘含量的基础上皮细胞的甲基化状态，据报道，这些细胞包含角膜上皮细胞的干细胞：我们发现，角蛋白12在这些细胞中处于高甲基化状态，这表明角蛋白12可能是当叶片基底细胞分化为角膜细胞的细胞时可能被脱甲基化的。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Corneal epithelial stem cells and differentiation

角膜上皮干细胞及其分化

DOI：
发表时间：
2007
期刊：
影响因子：
0
作者：
Shimizu N;Watanabe H;Kubota J;Wu J;Saito R;Yokoi T;Era T;Iwatsubo T;Watanabe T;Nishina S;Azuma N;Katada T;Nishina H.;木下茂
通讯作者：
木下茂

Different expression of angiogenesis-related factors between human cultivated corneal and oral epithelial sheets

DOI：
10.1016/j.exer.2006.02.015
发表时间：
2006-10-01
期刊：
EXPERIMENTAL EYE RESEARCH
影响因子：
3.4
作者：
Sekiyama, Eiichi;Nakamura, Takahiro;Kinoshita, Shigeru
通讯作者：
Kinoshita, Shigeru

Establishment of a cultivated human conjunctival epit helium as an alternative tissue source for autologous come al epithelial transplantation

建立培养的人结膜上皮氦作为自体上皮移植的替代组织来源

DOI：
发表时间：
2006
期刊：
Invest Ophthalmol Vis Sci 47
影响因子：
0
作者：
Tanioka;H.;Kawasaki;S.;Yamasaki;K.;Ang L.P.;Koizumi;N.;Nakamura;T.;Yokoi;N.;Komuro;A.;Inatomi;T.;Kinoshita;S
通讯作者：
S

Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation