ORIGIN AND MAINTENANCE OF THE OCULAR SURFACE EPITHELIA

眼表上皮的起源和维持

• 批准号: 8514617
• 负责人: DAVID CY BEEBE
• 金额: $ 28.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514617
• 关键词: Ablation; Address; Adhesions; Adult; Animals; Applications Grants; Binding; Blindness; Cell Adhesion Molecules; Cell Proliferation; Cell physiology; Cell-Cell Adhesion; Cells; Chemicals; Collaborations; Complement; Conjunctival Epithelium; Cornea; Corneal Stroma; Critical Pathways; Cytokine Signaling; Development; Differentiation Antigens; Ectoderm; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Exons; Eye; Eye diseases; Eyelash; Eyelid structure; Film; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Genome; Gland; Green Fluorescent Proteins; Hereditary Disease; Image; In Situ Hybridization; In Vitro; Inflammation; Injury; Knowledge; Label; Laboratories; Lacrimal gland structure; Lasers; Life; Limbus Corneae; Lipids; Maintenance; Mediating; Methods; Microarray Analysis; Microdissection; Mucous body substance; Mus; N-Cadherin; Pathway interactions; Pattern; Play; Property; Proteins; Publications; Reporter Genes; Reporting; Research Personnel; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Pathway Gene; Site; Specific qualifier value; Stem cells; Stromal Cell-Derived Factor 1; Surface; System; Tamoxifen; Testing; Time; Tissue Differentiation; Tissues; To specify; Undifferentiated; Vision; Work; adult stem cell; bone morphogenetic protein receptors; cell type; conjunctiva; corneal epithelial stem cells; corneal epithelium; eye dryness; immunocytochemistry; in vitro Assay; in vivo; induced pluripotent stem cell; injured; irritation; lacrimal; limbal; meibomian gland; neovascularization; notch protein; novel; ocular surface; prevent; recombinase; selective expression; stem; stem cell biology; stem cell niche; tool; transcription factor

DESCRIPTION (provided by applicant): The tissues on the surface of the eye and the secretory glands that are derived from these tissues are essential for vision. The lacrimal, Meibomian and conjunctival mucus glands secrete the components of the tear film. Insufficient function of any of these glands leads to dry eye disease. The corneal and limbal epithelia are responsible for maintaining the refractive surface of the eye. Deficiencies in the differentiation f the corneal epithelial cells or insufficient generation of corneal epithelial cells by the limbal sem cells leads to severe ocular irritation, inflammation, neovascularization of the corneal stroma and blindness. The aims of this proposal are 1) to identify the signaling systems that are responsible for the proper formation and function of the ocular surface epithelia and their derivatives during development and 2) to identify the signaling pathways that establish and maintain the limbal stem cells to provide functional corneal epithelial cells. The studies described in this proposal show that Pax6 and BMP signaling are key factors required for the formation and differentiation of all of the ocular surface epithelia. Laser microdissection and microarray analysis identified or confirmed four transcription factors that are early markers for the different ocular surface epithelia and are likely to play important roles in their differentiaton. Three of these factors depend on Pax6 for their expression. This proposal also describes a new method to genetically mark the limbal stem cells. At the same time, genes within these cells can be selectively deleted. This method will be used to inactivate critical pathways known to function in other adult stem cells. Three of these pathways, BMP and SDF-1 signaling and adhesion between niche and stem cells mediated by N-cadherin, have been shown by our collaborator on this project, Dr. Scheffer Tseng, to be involved in signaling between limbal stem and niche cells. The functions of these pathways will be tested in vivo by genetic ablation and confocal imaging. In each case, our vivo analyses will be complemented by in vitro genetic studies performed in Dr. Tseng's laboratory. This collaboration is possible because Dr. Tseng isolates limbal stem and adherent niche cells after overnight incubation. We will send eyes from our genetically-modified mice to Dr. Tseng by overnight courier. He will isolate the niche and stem cells and treat them with tamoxifen to delete the targeted genes. This collaboration will provide the most extensive analysis to date of the pathways that create and maintain the limbal stem cell niche. Information derived from both aims will be valuable for the replacement of injured or defective corneal and conjunctival epithelia using induced pluripotent cells or by "reprogramming" of other epithelial cell types.

描述（由申请人提供）：眼睛表面的组织以及源自这些组织的分泌腺对于视力至关重要。泪腺、睑板腺和结膜粘液腺分泌泪膜成分。这些腺体的功能不足会导致干眼病。角膜和角膜缘上皮负责维持眼睛的屈光表面。角膜上皮细胞分化缺陷或角膜缘干细胞生成角膜上皮细胞不足会导致严重的眼部刺激、炎症、角膜基质新生血管形成和失明。该提案的目的是：1）确定负责眼表上皮及其衍生物在发育过程中正确形成和功能的信号系统；2）确定建立和维持角膜缘干细胞以提供功能性角膜上皮细胞的信号传导途径。该提案中描述的研究表明 Pax6 和 BMP 信号传导是所有眼表上皮形成和分化所需的关键因素。激光显微切割和微阵列分析识别或证实了四种转录因子，它们是不同眼表上皮细胞的早期标记，并且可能在其分化中发挥重要作用。其中三个因子的表达依赖于 Pax6。该提案还描述了一种对角膜缘干细胞进行基因标记的新方法。同时，这些细胞内的基因可以被选择性删除。该方法将用于灭活已知在其他成体干细胞中起作用的关键途径。我们该项目的合作者 Scheffer Tseng 博士已证明，其中的三种途径，即 BMP 和 SDF-1 信号传导以及由 N-钙粘蛋白介导的微环境和干细胞之间的粘附，参与了角膜缘干细胞和微环境细胞之间的信号传导。这些途径的功能将通过基因消融和共聚焦成像在体内进行测试。在每种情况下，我们的体内分析都会得到曾博士实验室进行的体外遗传研究的补充。这种合作之所以成为可能，是因为曾博士在过夜孵育后分离出了角膜缘干细胞和贴壁微环境细胞。我们将通过隔夜快递将转基因小鼠的眼睛寄给曾博士。他将分离生态位和干细胞，并用他莫昔芬处理它们以删除目标基因。此次合作将为创建和维持角膜缘干细胞生态位的途径提供迄今为止最广泛的分析。从这两个目标获得的信息对于使用诱导多能细胞或通过“重新编程”其他上皮细胞类型来替换受损或有缺陷的角膜和结膜上皮来说将是有价值的。