Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis
Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis
复制标题
DOI：
10.1111/j.1600-0765.2010.01276.x
复制
发表时间：
2010-10-01
影响因子：
3.5
通讯作者：
Tachikawa, T.
中科院分区：
文献类型：
--
作者：
Hayashi, Y.;Matsunaga, T.;Tachikawa, T.
背景与目的：牙龈交界处的牙釉质附着在牙釉质上。结界上皮的附着机制已被组织学研究，但结界上皮的分子功能尚未阐明。本研究的目的是对结界上皮的基因表达进行综合分析，并寻找结界上皮的特异性遗传标记。材料与方法：采用激光显微解剖和微阵列分析技术对小鼠口腔粘膜上皮和结膜上皮中表达的基因进行综合分析。为了从这些组织中提取高质量的RNA，我们使用改进的薄膜法制作了冷冻切片。通过实时荧光定量PCR和免疫组织化学方法确认所选基因的差异表达。结果：改进的方法产生了足够质量的RNA用于微阵列分析。微阵列分析结果显示，与口腔牙龈上皮相比，841个基因在结膜上皮中表达上调，其中5个基因在结膜上皮中表达上调超过50倍。这5个基因分别是：分泌性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 （my16）和内质网蛋白29 （Erp29）。特别是，通过实时PCR，结膜上皮中Slpi的表达量比口腔牙龈上皮高约100倍。免疫组化结果显示，Slpi蛋白在结膜上皮中高表达。结论：我们建立了一种适合提取高质量RNA的新鲜冷冻组织切片的方法。我们确定Slpi在连接上皮中特征性表达。我们的研究结果在分析结膜上皮基因表达方面提供了实质性的进展。
Background and Objective: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.Material and Methods: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.Results: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.Conclusion: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.