Department of Periodontology, Showa University School of Dentistry, Ohta-ku, Tokyo, Japan.
J Periodontal Res. 2010 Oct;45(5):618-25. doi: 10.1111/j.1600-0765.2010.01276.x. Epub 2010 Jun 10.
The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium.
A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry.
The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium.
We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.
结合上皮附着于牙釉质和龈沟上皮的交界处。结合上皮的附着机制已通过组织学进行了研究，但结合上皮的分子功能尚未阐明。本研究旨在对结合上皮中的基因表达进行全面分析，并寻找结合上皮的特异性遗传标记。
使用激光显微切割和微阵列分析对小鼠结合上皮和口腔龈上皮中的基因表达进行了全面分析。为了从这些组织中提取高质量的 RNA，我们使用改良的膜法制作了冷冻切片。通过定量实时 PCR 和免疫组织化学验证了所选基因差异表达的确认。
改良的方法产生了足够质量用于微阵列分析的 RNA。微阵列分析的结果表明，与口腔龈上皮相比，841 个基因在结合上皮中上调，其中 5 个在结合上皮中上调超过 50 倍。这五个基因是分泌白细胞蛋白酶抑制剂（Slpi）、角蛋白 17（Krt17）、膜联蛋白 A1（Anxa1）、肌球蛋白轻肽 6（Myl6）和内质网蛋白 29（Erp29）。特别是，实时 PCR 显示结合上皮中 Slpi 的表达比口腔龈上皮高约 100 倍。此外，免疫组织化学表明 Slpi 蛋白在结合上皮中高度表达。
我们开发了一种适合提取高质量 RNA 的新鲜冷冻组织切片生成方法。我们确定 Slpi 是结合上皮的特征性表达。我们的结果为结合上皮中的基因表达分析提供了重大进展。
