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

我们采用随机cDNA测序法研究了人角膜上皮mRNA的组成。人类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）、钙调素、α -烯醇化酶、人脂肪mRNA和一个新基因的转录本。在角膜和结膜上皮以及其他粘膜上皮（阴道、食管、胃、小肠和结肠）和表皮中，研究了最丰富的转录因子apoJ mRNA和蛋白的表达。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。apojmrna在角膜上皮各层均有表达，以基底层居多。ApoJ蛋白定位于角膜和结膜上皮的顶端细胞层，以及阴道、食管、胃、小肠和结肠等各种粘膜上皮，但不在表皮中。这些结果表明，apoJ可能在泪眼表面界面起重要的生理作用。我们克隆了该新基因的全长cDNA。该基因与小鼠角蛋白12高度同源，表明该基因是人类角膜特异性角蛋白K12。结膜上皮细胞cDNA文库构建方面，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.