Analysis of the stress response in oral lesions by using Nrf2 gene knockout mouse

本研究的目的是分析转录因子Nrf2与口腔疾病的关系。作为基础研究，我们研究了Nrf2在抗氧化应激中的作用，并证实了氧化应激蛋白，如血红素加氧酶、过氧化物酶MSP23、A170等都受Nrf2的调控。我们还发现MSP23被BHA处理诱导。通过这些实验，我们发现Nrf2小鼠的表型是上颌切牙的脱色。我们分析了这一现象，以了解Nrf2在口腔疾病中的意义。首先将切牙样品用乙醇固定，用临界点干燥法脱水，然后用扫描电镜观察切牙表面。同、异、野生型小鼠之间无差异。用能量消耗微量X射线分析仪对钙、磷、铁离子含量进行了定量分析，发现小鼠间存在显著性差异。基因敲除小鼠体内铁离子含量明显下降，但钙和磷含量无差异。我们估计铁离子与nrf2基因破坏引起的口腔疾病的关联。为了解铁在切牙脱色过程中的作用，我们采用原位杂交、免疫组织化学和铁染色等方法研究了铁蛋白和铁离子的定位。结果发现成釉细胞内铁沉积异常，成釉细胞受损。提示Nrf2可能通过异常铁沉积而影响口腔疾病的发生。

The purpose of this study is to analyze how the transcription factor Nrf2 is associated with oral diseases. As fundamental study, we investigated the role of Nrf2 against oxidative stress and confirmed that the oxidative stress proteins, such as hemeoxygenase, peroxireoxin MSP23, A170, are under control of Nrf2 regulation. We also found MSP23 was induced by BHA treatment. Through these experiments we found the phenotype of Nrf2 mice was the decolorization of the maxillary incisor. We analyzed this phenomenon to know the significance of Nrf2 in oral desease. At first the incisor samples were fixed in ethanol and dehydraded by critical point drying method, and we observed the suface of the incisors with scanning electron microscope. There was no difference among the homo, hetero and wild type mice. We quantified the Ca, P, and Fe ion content with energy disparative micro X ray analysis and found there were significant difference among the mice. Iron ion content decreased significantly in knockout mice, though Ca and P content was not different. We estimated the association the iron ion and oral disease caused by nrf2 gene disruption. To know how the iron acted on the incisor decolorization, we investigated the ferritin and iron ion localization by using in situ hybridization, immunohistochemistry and iron staining. We found there were abnormal iron deposition and the ameloblast was damaged. Our results suggested Nrf2 affected on the oral diseases through abnormal iron deposition.