EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

本文提出的主要工作目标是：（1）检查 角膜内皮短链胶原的结构 （CESCC），其由兔角膜内皮细胞合成， （2）建立结构演化关系 在编码这种胶原蛋白的基因和编码 其他短链胶原蛋白（IX、X、XII型）; 3）检查 CESCC分子的组装和分泌在高度 组织化的角膜细胞外基质; 4）检查角膜的细胞外基质。 CESCC在胚胎期角膜中的表达 发育;和5）分析调节表达的 后胶原性视网膜脱离体外模型中的CESCC分子 层（角膜后纤维膜）。 为了实现这些目标，我们将使用DNA 克隆/测序和蛋白质化学。 cDNA编码 将从兔角膜构建CESCC分子 内皮细胞RNA。 该分子的一级结构为 通过分离的克隆的核苷酸序列分析确定。 编码CESCC分子的基因将分离自 兔基因组文库。 合成肽将由以下核苷酸序列制备： 兔cDNA 特异性抗体将产生针对这些 缩氨酸 这种抗体与特异性DNA探针一起， 将用于检查CESCC分子的合成， 其基因在角膜发育中的表达以及在 培养的内皮细胞。 这项研究应提供信息， 关于一种新型短链的结构和功能作用 角膜胶原及其调节机制 合成. 这些信息将证明是非常宝贵的， 了解正常细胞外基质的组装 角膜的改变和疾病引起的改变。

The primary goals of the work proposed here are: 1) to examine the structure of the corneal endothelial short-chain collagen (CESCC) which is synthesized by rabbit corneal endothelial cells, and 2) to establish the structural and evolutionary relationship between the gene(s) encoding this collagen and genes that code for other short-chain collagens (Types IX, X, XII); 3) to examine the assembly and secretion of the CESCC molecule in the highly organized extracellular matrices of the cornea; 4) to examine the expression of the CESCC in the cornea during embryonic development; and 5) to analyze the modulated expression of the CESCC molecule in an in vitro model of posterior collagenous layer (retrocorneal fibrous membrane). To accomplish these goals, we will use a combination of DNA cloning/sequencing and protein chemistry. cDNA coding for the CESCC molecule will be constructed from rabbit corneal endothelial cell RNA. Primary structure of this molecule will be determined by nucleotide sequence analysis of isolated clones. The gene(s) coding of the CESCC molecule will be isolated from rabbit genomic libraries. Synthetic peptides will be made from the nucleotide sequence of rabbit cDNA. Specific antibodies will be generated against these peptides. Such antibodies, together with specific DNA probes, will be used to examine the synthesis of the CESCC molecule and the expression of its gene(s) in corneal development as well as in cultured endothelial cells. The study should provide information about the structure and functional role of a novel short-chain collagen of the cornea and the mechanisms regulating its synthesis. Such information should prove invaluable for understanding the assembly of the normal extracellular matrices of the cornea and alterations due to disease.