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

本研究采用随机基因测序的方法，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，最丰富的转录本是载脂蛋白J(ApoJ)、钙周期蛋白、α-烯醇化酶、人类脂肪mRNA和一个新基因的转录本。研究了最丰富的转录体apoJ在角膜和结膜上皮以及其他粘膜上皮(阴道、食道、胃、小肠和结肠)和表皮中的表达。ApoJ mRNA在角膜、结膜、阴道和食道上皮细胞中均有表达，而在表皮角质形成细胞中未见表达。ApoJ信使核糖核酸表达于角膜上皮各层，以基底层表达最强。ApoJ蛋白定位于角膜和结膜上皮的顶层细胞层，以及阴道、食管胃、小肠和结肠等多种粘膜上皮细胞，而在表皮中不表达。这些结果表明apoJ可能在泪眼表面起着重要的生理作用。该基因含有与小鼠角蛋白12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本采集方法。

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.