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Gene expression analysis of human corneal and conjunctival epithelium by random cDNA sequencing

通过随机 cDNA 测序分析人角膜和结膜上皮的基因表达

基本信息

批准号：
06454500
负责人：
KINOSHITA Shigeru
金额：
$ 4.29万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454500/
关键词：
human corneal epithelium cDNA library random sequence gene expression known gene unknown gene in vivo cDNAライブラリー

项目摘要

We studied the composition of mRNA in human corneal epithelium by the random cDNA sequencing. The expression profile of active genes in human corneal epithelium showed that the most adundant transcripts represented those of genes for apolipoprotein J (apoJ), calcyclin, alpha-enolase, human adipose mRNA and a novel gene.The expression of both mRNA and protein of the most abundant trascripts, apoJ was investigated in corneal and conjunctival epithelium, as well as other mucosal epithelia (vagina, esophagus, stomach, small intestine, and colon) and epidermis. ApoJ mRNA transcripts was detected in corneal, conjunctival, vaginal and esophageal epithelium, but not in epidermal keratinocytes. ApoJ mRNA localized in all layrs of corneal epithelium, prominently in basal layr. ApoJ protein localized in the apical cell layrs of corneal and conjunctival epithelium, as well as various mucosal epithelia including vagina, esophagus stomach, small intestine and colon, but not in epidermis. These results indicated that apoJ may play a physiologically important role at the tear-ocular surface interface.We cloned the full lenght cDNA of the novel gene. The cDNA contained ORF highly homologous to mouse keratin 12, indicating that this gene is human cornea-specific keratin K12.Regarding the cDNA library construction of conjunctival epithelial cells, we are studying the sample collection method without contamination by blood cells or subepithelial cells.

采用随机cDNA序列测定方法，研究了人角膜上皮细胞中mRNA的组成。人角膜上皮中活性基因的表达谱显示，载脂蛋白J（apoJ）、钙周期素、α-烯醇化酶、人脂肪mRNA和一个新基因的转录本最丰富，其中apoJ的mRNA和蛋白质在角膜、结膜上皮及其他粘膜上皮中的表达最丰富（阴道、食道、胃、小肠和结肠）和表皮。在角膜、结膜、阴道和食管上皮中检测到ApoJ mRNA转录物，但在表皮角质形成细胞中未检测到。ApoJ mRNA在角膜上皮各层均有表达，以基底层表达最多。ApoJ蛋白定位于角膜、结膜上皮及阴道、食管、胃、小肠、结肠等多种粘膜上皮的顶端细胞层，而不定位于表皮。这些结果表明apoJ在泪液-眼表界面可能起着重要的生理作用。该cDNA含有与小鼠角蛋白K12高度同源的ORF，表明该基因是人角膜特异性角蛋白K12。关于结膜上皮细胞cDNA文库的构建，我们正在研究不受血细胞或上皮下细胞污染的样品收集方法。