Hu Shijia, Parker Joel, Wright John Timothy
Pediatric Dentistry, University of North Carolina, Chapel Hill, North Carolina, United States of America; Oral Biology Curriculum, University of North Carolina, Chapel Hill, North Carolina, United States of America.
Cancer Genetics, University of North Carolina, Chapel Hill, North Carolina, United States of America.
PLoS One. 2015 Apr 7;10(4):e0124801. doi: 10.1371/journal.pone.0124801. eCollection 2015.
The goal of the study was to characterize the transcriptome profiles of human ameloblasts and odontoblasts, evaluate molecular pathways and advance our knowledge of the human "dentome". Laser capture microdissection was used to isolate odontoblasts and ameloblasts from human tooth buds (15-20week gestational age) from 4 fetuses. RNA was examined using Agilent 41k whole genome arrays at 2 different stages of enamel formation, presecretory and secretory. Probe detection was considered against the array negative control to control for background noise. Differential expression was examined using Significance Analysis of Microarrays (SAM) 4.0 between different cell types and developmental stages with a false discovery rate of 20%. Pathway analysis was conducted using Ingenuity Pathway Analysis software. We found that during primary tooth formation, odontoblasts expressed 14,802 genes, presecretory ameloblasts 15,179 genes and secretory ameloblasts 14,526 genes. Genes known to be active during tooth development for each cell type (eg COL1A1, AMELX) were shown to be expressed by our approach. Exploring further into the list of differentially expressed genes between the motile odontoblasts and non-motile presecretory ameloblasts we found several genes of interest that could be involved in cell movement (FN1, LUM, ASTN1). Furthermore, our analysis indicated that the Phospholipase C and ERK5 pathways, that are important for cell movement, were activated in the motile odontoblasts. In addition our pathway analysis identified WNT3A and TGFB1 as important upstream contributors. Recent studies implicate these genes in the development of Schimke immuno-osseous dysplasia. The utility of laser capture microdissection can be a valuable tool in the examination of specific tissues or cell populations present in human tooth buds. Advancing our knowledge of the human dentome and related molecular pathways provides new insights into the complex mechanisms regulating odontogenesis and biomineralization. This knowledge could prove useful in future studies of odontogenic related pathologies.
本研究的目的是描绘人成釉细胞和成牙本质细胞的转录组图谱，评估分子途径并增进我们对人类“牙基因组”的了解。采用激光捕获显微切割技术从4例胎儿的人牙胚（孕龄15 - 20周）中分离成牙本质细胞和成釉细胞。在釉质形成的两个不同阶段，即分泌前期和分泌期，使用安捷伦41k全基因组芯片检测RNA。针对芯片阴性对照进行探针检测以控制背景噪声。使用微阵列显著性分析（SAM）4.0软件在不同细胞类型和发育阶段之间检测差异表达，错误发现率为20%。使用Ingenuity Pathway Analysis软件进行通路分析。我们发现，在乳牙形成过程中，成牙本质细胞表达14,802个基因，分泌前期成釉细胞表达15,179个基因，分泌期成釉细胞表达14,526个基因。我们的方法显示每种细胞类型在牙齿发育过程中已知活跃的基因（如COL1A1、AMELX）均有表达。进一步探究活动的成牙本质细胞和不活动的分泌前期成釉细胞之间差异表达基因列表，我们发现了几个可能参与细胞运动的感兴趣基因（FN1、LUM、ASTN1）。此外，我们的分析表明，对细胞运动很重要的磷脂酶C和ERK5通路在活动的成牙本质细胞中被激活。另外，我们的通路分析确定WNT3A和TGFB1为重要的上游贡献因子。最近的研究表明这些基因与施密克免疫骨发育不良的发生有关。激光捕获显微切割技术在检查人牙胚中存在的特定组织或细胞群体方面可能是一种有价值的工具。增进我们对人类牙基因组及相关分子途径的了解为调控牙发生和生物矿化的复杂机制提供了新的见解。这些知识可能在未来牙源性相关病理学研究中证明有用。
