Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium
Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium
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DOI：
10.1167/iovs.06-0216
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发表时间：
2006-09-01
影响因子：
4.4
通讯作者：
Piatigorsky, Joram
中科院分区：
文献类型：
--
作者：
Adachi, Wakako;Ulanovsky, Hagit;Piatigorsky, Joram
目的.鉴定在富含干细胞的大鼠角膜缘上皮中优先表达的基因。采用显微分离法分离6周龄大鼠角膜缘和中央角膜上皮细胞。构建基因表达系列分析（SAGE）文库并进行分析，采用常规方法进行原位杂交、逆转录-聚合酶链反应（RT-PCR）和cDNA克隆。大鼠角膜缘和中央角膜上皮SAGE文库分别由41，894和40，691个标签组成。注释后，这被减少到759个特定的角膜缘库和844个特定的中央角膜库的转录本; 2292个转录本重叠。编码具有代谢功能的蛋白质的转录物包括两个文库中的主要功能类别。原位杂交和/或RT-PCR结果的12个最丰富的，高度富集的成绩单在角膜缘上皮的SAGE数据一般协议，并表明，这些蛋白质也表达在结膜上皮。令人感兴趣的富含边缘的转录物编码WDNM 1样蛋白（类似于WDNM 1/Expi，一种假定的分泌蛋白酶和转移抑制剂）、间皮素（一种癌症标记物）、marapsin（一种胰蛋白酶样丝氨酸蛋白酶，可控制细胞生长和迁移）、K4和K15（均为细胞角蛋白）以及跨膜四结构域亚家族A成员8B。克隆了WDNM 1样蛋白，并证实其为四硫键核心蛋白家族的成员。SAGE结果扩展了啮齿类动物角膜中表达的基因数据库，并表明在角膜缘上皮中优先表达的几个基因与细胞增殖和迁移之间存在关联。
PURPOSE. To identify genes preferentially expressed in the stem-cell-rich limbal epithelium of the rat cornea.METHODS. The limbal and central corneal epithelial cells of 6-week-old rats were isolated by microdissection. Serial analysis of gene expression ( SAGE) libraries were constructed and analyzed, and in situ hybridization, reverse transcription-polymerase chain reaction ( RT-PCR) and cDNA cloning were conducted by conventional procedures.RESULTS. The rat limbal and central corneal epithelial SAGE libraries consisted of 41,894 and 40,691 tags, respectively. After annotation, this was reduced to 759 transcripts specific for the limbal library and 844 transcripts specific for the central corneal library; 2292 transcripts overlapped. Transcripts encoding proteins with metabolic functions comprised the major functional category in both libraries. In situ hybridization and/or RT-PCR results of 12 of the most abundant, highly enriched transcripts in the limbal epithelium were in general agreement with the SAGE data and showed that these proteins are also expressed in the conjunctival epithelium. Interesting limbal-enriched transcripts encode WDNM1-like protein ( similar to WDNM1/Expi, a putative secreted proteinase and inhibitor of metastasis), mesothelin ( a cancer marker), marapsin ( a trypsin-like serine protease that may control cell growth and migration), K4 and K15 ( both cytokeratins), and membrane-spanning four-domain subfamily A member 8B. WDNM1-like protein was cloned and confirmed as a member of the four-disulfide core family.CONCLUSIONS. The SAGE results extend the database of genes expressed in the rodent cornea and suggest an association between several genes preferentially expressed in the limbal epithelium with cellular proliferation and migration.