DIFFERENTIAL GENE EXPRESSION IN CILIARY EPITHELIUM

眼睫状体上皮是房水形成的部位。 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学上的区域差异 这是生物化学、生理和分子差异的基础。《长河》 这项提案的术语目标是在分子水平上表征 无色(NPE)与有色(NPE)的区别 (PE)正常和病变眼组织中的睫状体上皮细胞。 有关睫状上皮细胞特异性基因的信息可能阐明关键 人类病变睫状体上皮可能受损的功能 细胞。具体目标如下： A)构建消减NPE和PE消减文库 利用NPE和PE的Poly A+mRNA构建定向cDNA文库 细胞分别构建在Uni-ZAP XR载体中。使用互补性 杂交可以获得丰富的NPE和PE特异性克隆。 我们将免疫筛选牛和/或人的表达基因 用lambda-gtll和Uni-ZAP XR载体构建的文库 鉴别NPE和PE细胞的单抗和多克隆抗体 抗原性的。 B)通过限制性内切酶鉴定NPE和PE特异的DNA序列 核酸内切酶图谱和DNA序列分析。我们已经隔离了 并对几个牛的cdna克隆进行了部分鉴定。 睫状体上皮细胞cDNA文库。这些牛的cdna克隆， 显示细胞和区域特异性，将被用来分离和 描述相应的人类全长克隆的特征。 C)评估NPE和PE特有的基因表达水平 睫状体上皮原位杂交克隆。 识别编码已知蛋白质的细胞特异性克隆应 允许将抗体设计为技术工具，用于 蛋白质的生物发生、组装和结构分析。我们将进行化验 维甲酸和转化生长因子-β在血管紧张素转换酶中的作用 利用NPE和PE细胞维持分化的基因表达。在……里面 特别是细胞视黄醛结合蛋白的表达 (CRALBP)可以提供维甲酸类物质如何 输送到房水中。 D)睫状体上皮基因在病理性眼部疾病中的表达。 我们将鉴定和检测以下克隆的表达水平 已经改变了眼睫状体上皮细胞的基因表达 病理病例(如青光眼、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).