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对抗氧化应激的作用，并证实了氧化应激蛋白，如血红素加氧酶、过氧酶MSP23、A170，受Nrf2调控。我们还发现BHA诱导了MSP23的表达。通过这些实验，我们发现Nrf2小鼠的表型是上颌切牙的脱色。我们对这一现象进行了分析，以了解NRF2在口腔疾病中的意义。首先将切牙标本用乙醇固定，用临界点干燥法脱水，用扫描电子显微镜观察切牙表面。同系小鼠、异种系小鼠和野生型小鼠之间无差异。我们用能量差显微X射线分析对钙、磷、铁离子的含量进行了定量，发现小鼠之间有显著差异。基因敲除小鼠体内铁离子含量显著降低，但钙、磷含量无明显差异。我们估计了Nrf2基因突变引起的铁离子与口腔疾病之间的关系。为了了解铁在切牙脱色中的作用，我们用原位杂交、免疫组织化学和铁染色的方法研究了铁蛋白和铁离子的定位。我们发现有异常的铁沉积，成釉细胞受损。提示Nrf2可能通过异常铁沉积作用于口腔疾病。

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.