DIFFERENTIAL GENE EXPRESSION IN CILIARY EPITHELIUM

眼睫状体上皮是眼房水形成的部位， 哺乳动物的眼睛 汇编的证据表明，沿着眼睛 睫状体上皮存在解剖区域差异， 构成了生物化学、生理学和分子差异的基础。 长 本提案的长期目标是在分子水平上进行表征 非色素性（NPE）和色素性（NPE） (PE)正常和患病眼组织中的睫状上皮细胞。 睫状体上皮细胞特异性基因的信息可以阐明 可能在人类患病睫状上皮中受损的功能 细胞 具体目标如下： A）从两个细胞中制备消减NPE和PE cDNA文库 从NPE和PE的poly A+ mRNA构建定向cDNA文库 细胞分别在Uni-ZAP XR载体中。使用互补 通过杂交，我们可以获得NPE和PE特异性克隆的富集。 我们将免疫筛选牛表达和/或人表达cDNA 在具有一组DNA的IgGda-gtll和Uni-ZAP XR载体中构建的文库 区分NPE和PE细胞的单克隆和多克隆抗体 抗原性 B）通过限制性内切酶对NPE和PE特异性DNA序列的表征 核酸内切酶作图和DNA序列分析。 我们已经隔离了 并部分表征了来自牛的几个牛cDNA克隆， 睫状体上皮cDNA文库。 这些牛cDNA克隆， 显示细胞和区域特异性，将用于分离和 表征相应的人类全长克隆。 C）评估NPE和PE特异性免疫应答的基因表达水平。 通过睫状上皮中的原位杂交克隆。 鉴定编码已知蛋白质的细胞特异性克隆， 允许抗体被设计成作为技术工具， 分析蛋白质的生物合成、组装和结构。 我们将分析 类维生素A和转化生长因子-β（TGF-β）在 使用NPE和PE细胞维持分化的基因表达。 在 特别是细胞视黄醇结合蛋白的表达 这些细胞中的CRALBP可以提供关于类维生素A是如何在细胞中表达的信息。 被输送到房水中。 D）睫状上皮基因在病理性眼病中的表达。 我们将鉴定和检测cDNA克隆的表达水平， 改变了眼睫状上皮的基因表达， 病理性病例（即青光眼、白内障或眼部炎症）。

The ocular ciliary epithelium is the site of aqueous humor formation in the mammalian eye. Compiled evidence indicates that along the ocular ciliary epithelium there are anatomical regional differences that underlie biochemical, physiological and molecular differences. The long term objective of this proposal is to characterize at the molecular level the differentiated properties of the nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells in normal and diseased ocular tissue. Information on ciliary epithelial cell-specific genes may elucidate key functions that may be impaired in human diseased ciliary epithelial cells. The specific aims are as follows: A) Preparation of subtractive NPE and PE cDNA libraries from two directional cDNA libraries constructed from poly A+ mRNA of NPE and PE cells respectively in Uni-ZAP XR vectors. using complementary hybridization we can obtain an enrichment of NPE and PE-specific clones. We will immunoscreen bovine expression and/or human expression cDNA libraries constructed in lambda-gtll and Uni-ZAP XR vectors with a group of monoclonal and polyclonal antibodies distinguishing NPE and PE cells antigenically. B) Characterization of NPE and PE-specific DNA sequences by restriction endonuclease mapping and DNA sequence analysis. We have already isolated and partially characterized several bovine cDNA clones from bovine ciliary epithelial cDNA libraries. These bovine cDNA clones, which display cellular and regional specificity, will be used to isolate and characterize the corresponding human full length clones. C) Assessing the level of gene expression for the NPE and PE-specific clones by in situ hybridization in the ciliary epithelium. Identification of cell-specific clones encoding known proteins should allow antibodies to be designed to function as technical tools for the analysis of protein biogenesis, assembly and structure. We will assay the role of retinoids and transforming growth factor-beta (TGF-beta) in maintaining differentiated gene expression using NPE and PE cells.. In particular the expression of the cellular retinaldehyde-binding protein (CRALBP) in these cells could provide information on how retinoids are transported into the aqueous humor. D) Expression of ciliary epithelial genes in pathological eye conditions. We will identify and examine the level of expression of cDNA clones which have altered gene expression in the ocular ciliary epithelium of pathological cases (i.e. glaucoma, cataracts or ocular inflammation).