Analysis of stress defense mechanism against oral cancer by using A170 gene knockout mouse

在这项研究中，我们重点研究了氧化应激蛋白A170，它被认为在口腔颌面部病变中发挥着重要作用，因为它与癌基因和成骨细胞分化有关。我们计划1)构建A170基因敲除小鼠，2)从临床和基本角度分析包括A170在内的氧化应激蛋白的应激反应。1)关于基因敲除小鼠的构建，我们进行了以下步骤。我们从BAC/129Sv小鼠基因组文库中分离了A170的基因组克隆，并对其基因进行了鉴定。在定位后，我们设计了A170-空的靶向载体。利用电穿孔技术将携带该载体的修饰基因导入ES细胞。将转基因细胞注射到C57/B6N来源的胚胎囊胚中，然后移植到代孕母亲体内。嵌合体的产生具有高刺鼠百分比的外套，这表明接受基因突变。然后将这些嵌合小鼠与CS7/B6小鼠交配。含有豚鼠皮毛的F1后代表明是生殖系遗传。经FCR鉴定证实为生殖系传递。2)在构建基因敲除小鼠的过程中，我们用大鼠研究了应激剂对A170的诱导作用。我们还研究了过氧化还蛋白I(Prx I=MSP23)和其他应激诱导蛋白的表达，作为A170基因敲除小鼠分析的基础。此外，我们还从临床样本中调查了口腔癌的表达水平。

In this study we focused on the oxidative stress protein A170 which was estimated to play a important role in oral and maxillofacial lesions because it was associated with the oncogene and osteoblast differentiation. We planed 1) to construct A170 gene knockout mouse and 2) to analyze the stress : response of the oxidative stress proteins including A170 from clinical and basic viewpoints.1) About knockout mouse construction we carried out the following procedures. We isolated the genomic clone and characterized the gene of A170 from BAC/129Sv mouse genome library. After mapping we designed the A170-null targeting vector. The modified gene with the vector was introduced into the ES cells by electroporation. Genetically altered cells were injected into embryonic blastocysts derived from C57/B6N and then implanted into a surrogate mother. Chimeras were generated with a high agouti percentage coat which indicated acceptance of the gene mutation. These chimeric mice were then mated with CS7/B6 mice. The F1 offspring containing the agouti coat indicated germline transmission. The germline transmission was confirmed by FCR. Finally we mated Flmouse and knock out mouse (F2) construction was completed.2) While knockout mouse construction we investigated the induction of A170 by stress agents using the rat. We also investigated Peroxiredoxin I (Prx I = MSP23) and the other stress inducible protein expression as a foundation for A170 knockout-mouse analysis. Moreover, we also investigated the expression level of oral cancer from clinical samples.