Dota A, Nishida K, Adachi W, Nakamura T, Koizumi N, Kawamoto S, Okubo K, Kinoshita S
Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan.
Exp Eye Res. 2001 Mar;72(3):235-41. doi: 10.1006/exer.2000.0946.
The purpose of this study was to describe the quantitatively and qualitatively genes expressed in in vivo human conjunctival epithelium. A cDNA library was created from human conjunctival epithelial cells obtained from 38 normal eyes by brush cytology. Poly A(+)RNA isolated from these cells was used as a template for cDNA synthesis by the vector-priming method. A 3'-directed cDNA library consisting of the cDNA moiety covering the poly A(+)RNA tail through the nearest Mbo I site was constructed and transformed into Escherichia coli. Inserts in 933 randomly chosen clones were sequenced. The resulting sequences were compared to determine frequency of appearance in the library, and to establish an expression profile of genes in the conjunctival epithelium. The sequences were sent to GenBank for gene identification. The result was an expression profile of active genes reflecting their relative abundance in the conjunctival epithelial mRNA population. The expression profile of human conjunctival epithelium showed that the most abundant gene transcript in human conjunctival epithelium was that for cytokeratin 13. Altogether 102 genes were found to be very active, including beta-2 microglobulin, lipocortin I and insulin-like growth factor binding protein-3. The gene expression profile of the conjunctival epithelium reflects the unique properties and functions of this tissue. Comparison of this expression profile with that obtained from corneal epithelium discloses clear differences and helps us better understand the physiology and pathophysiology of the ocular surface in humans.
本研究的目的是描述体内人结膜上皮中定量和定性表达的基因。通过刷片细胞学从38只正常眼睛获取人结膜上皮细胞，构建了一个cDNA文库。从这些细胞中分离的聚腺苷酸（+）RNA用作载体引发法合成cDNA的模板。构建了一个3'定向cDNA文库，其由覆盖聚腺苷酸（+）RNA尾部直至最近的Mbo I位点的cDNA部分组成，并转化到大肠杆菌中。对933个随机选择的克隆中的插入片段进行测序。比较所得序列以确定文库中出现的频率，并建立结膜上皮中基因的表达谱。将这些序列发送到GenBank进行基因鉴定。结果是反映了它们在结膜上皮mRNA群体中相对丰度的活性基因表达谱。人结膜上皮的表达谱表明，人结膜上皮中最丰富的基因转录本是细胞角蛋白13的转录本。总共发现102个基因非常活跃，包括β-2微球蛋白、脂皮质蛋白I和胰岛素样生长因子结合蛋白-3。结膜上皮的基因表达谱反映了该组织的独特特性和功能。将该表达谱与从角膜上皮获得的表达谱进行比较，揭示了明显的差异，并有助于我们更好地理解人类眼表的生理学和病理生理学。
