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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390472/
• 关键词: keratin 12; methylation; CpG island; transcription factor; epigenetics; ゲノムメチル化; 転写制御; エピジェネティクス

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是在角膜上皮细胞中特异性表达的基因。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，我们评估了各种类型的人类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。令人惊讶且值得注意的是，传代培养的原代人角膜上皮细胞 (hCEC) 和永生化 hCEC 不表达角蛋白 12。然后，我们用去甲基化剂 5-氮杂胞苷和组蛋白脱乙酰酶抑制剂 TSA 处理永生化 hCEC，以观察角蛋白 12 的这种组织特异性基因表达是否涉及表观遗传基因调控 机制。随后我们发现这些细胞表达角蛋白12。接下来，我们进行亚硫酸氢盐测序分析来观察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在体内角膜上皮细胞中处于低甲基化状态，而其他角质形成细胞则表现出角蛋白12的高甲基化。此外，传代培养的原代hCEC（P6、P7和P8）和永生化hCEC表现出角蛋白12的高甲基化。然后我们检验了培养过程可能影响角蛋白12的假设。 基因组甲基化状态。我们对 hCEC 进行了从体内到 P6 的重复培养，并对这些细胞中的每一个进行亚硫酸氢盐测序分析。我们发现角蛋白12甲基化率随着传代次数的增加而逐渐增加。最后，我们评估了角膜缘基底上皮细胞的甲基化状态，据报道这些细胞含有角膜上皮细胞的干细胞：我们发现这些细胞中角蛋白12处于高甲基化状态，表明当角膜缘基底细胞分化为角膜上皮细胞时角蛋白12可能去甲基化。

项目成果

Corneal epithelial stem cells and differentiation

角膜上皮干细胞及其分化

• 发表时间: 2007
• 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

Connexin43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo

• DOI: 10.1167/iovs.07-0255
• 发表时间: 2008-01-01
• 期刊: INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
• 影响因子: 4.4
• 作者: Nakano, Yukiko;Oyamada, Masahito;Takamatsu, Tetsuro
• 通讯作者: Takamatsu, Tetsuro

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

• 发表时间: 2006
• 期刊: Invest Ophthalmol Vis Sci 47
• 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

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

Clusters of corneal epithelial m11 s reside ectopica]ly in human conjunctival epithelium

角膜上皮细胞簇异位存在于人结膜上皮中

• 发表时间: 2006
• 期刊: Invest Ophthalmol Vis Sci 47
• 作者: Kawasaki;S.;Tanioka;H.;Yamasald;K;Yokoi;N;Komum;A;Kmoshita;S
• 通讯作者: S