COLLAGEN GENE EXPRESSION IN THE CORNEA

角膜中胶原蛋白基因的表达

• 批准号: 2162104
• 负责人: ICHIRO NISHIMURA
• 金额: $ 12.01万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2162104
• 关键词: DNA footprinting; RNase protection assay; bioassay; chick embryo; collagen; corneal stroma; electron microscopy; extracellular matrix; fibroblasts; gene expression; immunochemistry; in situ hybridization; molecular cloning; nonmammalian vertebrate embryology; nucleic acid probes; nucleic acid sequence; tissue /cell culture; western blottings

The avian corneal primary stroma has a unique structure. It consists of orthogonally arranged layers of collagen fibrils, composed of type I and II collagens that are produced by corneal epithelial cells. The primary stroma contains also type IX collagen, at least as a transitory component, and my recent studies show that the corneal form of this unique extracellular matrix molecule is different from that which is expressed in cartilage. Whereas cartilage type IX molecules contain a large globular amino-terminal domain located in the perifibrillar matrix along collagen fibrils, this domain is absent in corneal type IX collagen molecules. I propose that analysis of this difference will provide novel and exciting clues to answer the general question of what mechanisms cells utilize to build unique extracellular matrices in different tissues from a common set of extracellular matrix genes. As a first step in this analysis, DNA cloning and sequencing techniques will be applied to characterize the structure of corneal type IX collagen. In addition, rotary shadowing electron microscopy, western blotting and immunoprecipitation will be used to characterize the corneal type IX collagen protein. Second, the differential expression of the two different type IX collagens, the corneal and cartilage forms, will be examined by means of RNase protection methods in different tissues such as cornea, sclera, lens, retina, notochord, neural tube epithelium and cartilage. Third, to precisely locate the cells expressing the two forms of type IX collagen, in situ hybridization studies will be performed using DNA probes containing cornea and cartilage specific type IX collagen nucleotide sequences. Finally, the al(IX) gene will be examined in experiments aimed at deciphering the molecular mechanisms involved in expression of the corneal form of type IX collagen.

鸟类角膜原代基质具有独特的结构。 它包括 正交排列的胶原纤维层，由I型和 角膜上皮细胞产生的II型胶原。 主 基质还含有IX型胶原，至少作为一种暂时的胶原， 成分，我最近的研究表明，角膜形式的这种 独特的细胞外基质分子不同于 在软骨中表达。 而软骨IX型分子含有 位于纤维周围基质中的大球状氨基末端结构域 沿着胶原纤维，该结构域在角膜IX型中不存在 胶原分子 我建议，对这种差异的分析将 提供了新颖而令人兴奋的线索来回答这个普遍的问题， 细胞利用的机制，以建立独特的细胞外基质， 不同的组织从一个共同的一套细胞外基质基因。 作为这种分析的第一步，DNA克隆和测序技术 将应用于表征角膜IX型的结构 胶原 此外，旋转阴影电子显微镜，西方 印迹和免疫沉淀将用于表征 角膜IX型胶原蛋白。 二、差异化表达 在两种不同的IX型胶原中，角膜和软骨形式， 将通过不同的RNase保护方法进行检查， 组织如角膜、巩膜、透镜、视网膜、脊索、神经管 上皮和软骨。 第三，精确定位细胞 表达两种形式的IX型胶原，原位杂交 研究将使用含有角膜和 软骨特异性IX型胶原蛋白核苷酸序列。 最后 al（IX）基因将在旨在破译 参与角膜型的表达的分子机制 IX胶原蛋白。