EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

这里提出的工作的主要目标是：1)检查 角膜内皮短链胶原的结构 (CESCC)是由兔角膜内皮细胞合成的， 2)建立结构与进化的关系 在编码这种胶原蛋白的基因(S)和编码这种胶原蛋白的基因之间 其他短链胶原蛋白(IX、X、XII型)；3)检查 CESCC分子在人体内的组装和分泌 有组织的角膜细胞外基质；4)检查 胚胎期角膜上皮细胞癌的表达 发展；以及5)分析该基因的调控表达 后胶原体外模型中CESCC分子的研究 层(角膜后纤维膜)。 为了实现这些目标，我们将使用DNA的组合 克隆/测序和蛋白质化学。编码该基因的c DNA CESCC分子将从兔角膜中构建 内皮细胞RNA。这个分子的一级结构将是 通过对分离克隆的核苷酸序列分析确定。 编码CESCC分子的基因(S)将从 兔基因组文库。 合成肽将由以下核苷酸序列制成 兔cDNA.将会产生针对这些病毒的特异性抗体 多肽。这样的抗体，加上特定的DNA探针， 将用于研究CESCC分子的合成和 其基因(S)在角膜发育过程中的表达 培养内皮细胞。这项研究应该提供信息 关于一种新型短链的结构和功能作用 角膜胶原蛋白及其调控机制 综合。这些信息应该证明是无价的 了解正常细胞外基质的组装 角膜和因疾病引起的变化。

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.